CN109072280B - Method for rapid antimicrobial sensitivity testing - Google Patents

Abstract

The present invention relates in part to methods and kits for rapidly determining antimicrobial susceptibility of a microorganism. The methods and kits use signaling agents that specifically or non-specifically bind to the surface of a microorganism. Preferably, the signalling agent has an amplifier group, such as a europium coordination complex.

Description

Method for rapid antimicrobial sensitivity testing

RELATED APPLICATIONS

The present application claims 2016 U.S. provisional patent application No. 62/281,698 filed on month 1 and day 21; U.S. provisional patent application nos. 62/298,821, filed 2016, 2, 23; U.S. provisional patent application No. 62/326,545, filed 4/22/2016; U.S. provisional patent application No. 62/338,376, filed 2016, 5, 18; U.S. provisional patent application No. 62/370,579, filed 2016, 8, 3; and 2016, 9/383,198. The contents of the aforementioned patent application are incorporated herein by reference in their entirety.

Background

Antimicrobial resistant microbial infections are associated with poor clinical outcomes including increased morbidity, mortality and health care costs in infected patients. The prevalence of these organisms in such facilities in the united states has steadily increased over the last 30 years. Phenotypic Antimicrobial Susceptibility Testing (AST) of microorganisms is important to inform physicians of the appropriate treatment regimen. Using existing methods, AST assays typically require a minimum of eight hours, which is an overnight process due to the rotation in many clinical microbiology laboratories. While awaiting measurement from current AST methods, patients are often administered broad spectrum antimicrobials that often have significant adverse effects on patient health and/or contribute to an increasing antimicrobial resistance epidemic. Furthermore, such time delays in obtaining accurate antimicrobial therapy information increase patient stays in the hospital, thereby increasing patient costs and inconvenience.

Accordingly, there is a need for a method of rapidly determining antimicrobial susceptibility to microbial infections. The method described herein is further advantageous because it addresses this need in a cost effective manner because it is compatible with existing assay hardware components.

Summary of The Invention

The present invention allows for the rapid determination of antibiotic susceptibility of microbial infections. The present invention is based, in part, on the surprising discovery of non-specific surface binding assays that provide accurate and rapid Antimicrobial Sensitivity Test (AST) assays in less than twelve hours-and, in particular, in 4 hours. The present invention ("Fast-AST") provides accurate results consistent with those obtained using the Clinical Laboratory Standards Institute (CLSI) reference method when tested with multiple antimicrobial agents and multiple microorganisms; however, the present invention takes significantly less time to obtain results than the CLSI method. Furthermore, the present invention accurately distinguishes between an antimicrobial MIC of a clinically relevant strain of a microorganism that is resistant to one or more antimicrobial agents and an antimicrobial MIC of a strain of the same microorganism that is susceptible to the antimicrobial agent. Furthermore, the invention may include signaling agents (e.g., europium compounds) that bind non-specifically, rather than specifically, to the microorganism (e.g., via chemically conserved groups or biochemically conserved binding sites on the microorganism), thereby extending the generality of the invention to any microorganism and allowing the appropriate treatment to be initiated without first identifying a particular infectious microorganism. Furthermore, the invention allows signal amplification, such that microorganisms can be detected rapidly at lower concentrations, e.g. from diluted cultures of microorganisms or via biological samples of patients. Furthermore, the present invention can use europium formulations as the chemical moiety, thereby extending the dynamic range of the method and allowing more accurate assays to be performed from a range of microbial samples. Finally, the present invention is compatible with existing equipment, thereby enabling rapid adoption in current clinical laboratories. Thus, the present invention can provide patients with an appropriate treatment regimen, i.e., a specific antimicrobial agent and at a specific dosage, in a substantially reduced amount of time and expense relative to standard methods. Thus, the present invention will improve patient outcomes, reduce hospital costs, and help reduce the further evolution of antimicrobial resistant microorganisms; thus, the present invention represents a significant breakthrough in the AST field.

One aspect of the invention is a method for determining antimicrobial sensitivity of a microorganism. The method comprises the following steps: incubating a liquid suspension of microorganisms in the presence of an antimicrobial agent and a signaling agent capable of binding to the surface of the microorganisms under conditions that promote growth of the microorganisms; separating microorganisms bound by the signaling agent from unbound signaling agent; and determining the level of signal associated with the microorganism as compared to one or more controls.

Another aspect of the invention is a method for determining antimicrobial sensitivity of a microorganism. The method comprises the following steps: incubating a liquid suspension of microorganisms in the presence of an antimicrobial agent under conditions that promote growth of the microorganisms; adding a signaling agent capable of binding to the surface of the microorganism; separating microorganisms bound by the signaling agent from unbound signaling agent; and determining the level of signal associated with the microorganism as compared to one or more controls.

Yet another aspect of the invention is a method for determining antimicrobial sensitivity of a microorganism. The method comprises the following steps: incubating a liquid suspension of microorganisms in a cartridge comprising a plurality of chambers, each chamber containing one or more antimicrobial agents, under conditions that promote growth of the microorganisms; adding a signaling agent capable of binding to the surface of the microorganism to the plurality of chambers; removing unbound signaling agent; and determining the level of signaling in the plurality of chambers as compared to one or more controls.

One aspect of the invention is a method for determining antimicrobial sensitivity of a microorganism. The method comprises

Incubating the microorganism in the presence of an antimicrobial agent and a signaling agent under conditions that promote growth of the microorganism, the signaling agent comprising a signal amplifier and one or more chemical moieties capable of non-specifically binding to the surface of the microorganism; separating microorganisms bound by the signaling agent from unbound signaling agent; and determining the level of signal associated with the microorganism as compared to one or more controls.

Another aspect of the invention is a method for determining antimicrobial sensitivity of a microorganism. The method comprises incubating the microorganism in the presence of an antimicrobial agent under conditions that promote growth of the microorganism; adding a signaling agent comprising a signal amplifier and one or more chemical moieties capable of non-specifically binding to the surface of the microorganism; separating microorganisms bound by the signaling agent from unbound signaling agent; and determining the level of signal associated with the microorganism as compared to one or more controls.

Yet another aspect of the invention is a kit for determining antimicrobial sensitivity of a microorganism. The kit comprises a signaling agent capable of binding to the surface of an intact target microorganism; a solution for incubating a sample containing a microorganism; and one or more agents for generating a signal from the signaling agent.

Any aspect or embodiment described herein may be combined with any other aspect or embodiment as disclosed herein. While the present disclosure has been described in conjunction with the detailed description thereof, the foregoing description is intended to illustrate and not limit the scope of the disclosure, which is defined by the scope of the appended claims. Other aspects, advantages, and modifications are within the scope of the following claims.

The patent and scientific literature referred to herein establishes knowledge available to those skilled in the art. All U.S. patents and published or unpublished U.S. patent applications cited herein are incorporated by reference. All published foreign patents and patent applications cited herein are hereby incorporated by reference. All other published references, documents, manuscripts and scientific literature cited herein are incorporated by reference.

Other features and advantages of the invention will be apparent from the accompanying drawings and from the following detailed description and claims.

Brief Description of Drawings

The above and further features will be more clearly understood from the following detailed description when taken in conjunction with the accompanying drawings. However, the drawings are for illustration purposes only; and are not intended to be limiting.

FIG. 1 is a schematic diagram showing the general steps of the present invention.

Fig. 2A-2D are diagrams and illustrations showing key features of aspects of the present invention ("fast-AST").

Fig. 3 is a schematic diagram comparing the steps required in currently used Antimicrobial Susceptibility Testing (AST) systems and aspects of the present invention.

Fig. 4 is a graph showing the time delay required to obtain results using the AST system currently in use (i.e. Vitek2 from biomerie ux).

FIG. 5 is a graph showing the clindamycin versus Staphylococcus aureus (S.aureus) ("Flashast" technique) using the present invention and standard overnight growth followed by Optical Density (OD) reading at 600nmStaphylococcus aureus) Comparative figures for Minimum Inhibitory Concentration (MIC) determination of (ATCC strain 29213). Data shown for "quick-AST" are from 5 minute points after the start of incubation with the test solution and represent the mean and standard deviation of 4 wells, with the median being for each assay type versus the control without antimicrobial agent.

FIG. 6 is a graph showing ceftazidime versus Pseudomonas aeruginosa (OD) using the present invention ("Rapid-AST" technique) and standard overnight growth followed by Optical Density (OD) reading at 600nmPseudomonas aeruginosa) Comparative figures for MIC determination of (ATCC strain 27853). Data shown represent the mean and standard deviation of four wells, with the median being the control without antimicrobial for each assay type.

FIG. 7 shows the results for two P.aeruginosa strains: (A)Pseudomonas aeruginosa) The strain is as follows: graph comparing MIC assays using the present invention ("fast-AST") for sensitive (ATCC strain 27853) and resistant (ATCC strain BAA-2108) strains. Data shown represent the average of four wells, with values for eachThe assay is relative to a control without the antimicrobial agent.

Figure 8 is a table summarizing the data from example 2. It compares MIC calls (calls) by the "Rapid-AST" technique with the standard overnight OD ₆₀₀ Those of the program. The data shown represents the average of four wells, with values for each assay type relative to a control without antimicrobial agent.

Fig. 9 shows two techniques: the present invention ("Rapid-AST" amplification technique) and the standard optical Density technique (OD) at 600nm ₆₀₀ ) S. staphylococcus aureus: (a)Staphylococcus aureus) Graph comparing concentrations (in CFU/ml).

Figure 10 is a graph showing MIC results for the present invention ("rapid-AST" method) compared to the Clinical Laboratory Standards Institute (CLSI) reference method for seven pathogenic bacterial species.

Figure 11 is a table identifying bacteria, antimicrobial agents, and signaling agents/chemical moieties used in example 4.

FIGS. 12A to 12C show Staphylococcus aureus (S.aureus)S. aureus) (FIG. 12A) and Klebsiella pneumoniae: (B)K.pneumonia) (FIG. 12B) Table of representative SensiTitre results. No indications for nitrofurantonin Staphylococcus aureus: (S. aureus) Experimental use the data of the present invention was used because only two holes were dedicated to the antimicrobial agent.

FIGS. 13A-13C are graphs showing the treatment of Staphylococcus aureus (S.aureus) with the antimicrobial agents oxacillin (FIG. 13A), vancomycin (FIG. 13B) and levofloxacin (FIG. 13C)S. aureus) Graph of the comparison between MIC results obtained by the present invention and CLSI reference method for clinical strains.

FIGS. 14A-14D are graphs showing ampicillin (FIG. 14A), ciprofloxacin (FIG. 14B), imipenem (FIG. 14C), and gentamicin (FIG. 14D) on E.coli (E.coli) ((E.coli)) for the antimicrobial agents ampicillin (FIG. 14A), ciprofloxacin (FIG. 14B), imipenem (FIG. 14C), and gentamicin (FIG. 14D)E. coli) Graph of the comparison between MIC results obtained by the present invention and CLSI reference method for clinical strains.

FIG. 15 shows the present invention ("FAST-AST") in Staphylococcus aureus: (S.aureus) ((R))S. aureus) On the same clinical species ofGraphs of MIC results similar to those obtained by the CLSI standard reference method were consistently generated over the course of one month.

FIGS. 16-23 are graphs comparing chemically sensitive Escherichia coli (E. Coli) (I.coli) with various antimicrobial agentsE. coli) Graph of the sensitivity of strain ("QC 25922") and clinically relevant antimicrobial resistant strain ("clinical"). The antimicrobial agent used was imipenem (fig. 16); ampicillin (fig. 17); ceftazidime (fig. 18); gentamicin (fig. 19); levofloxacin (figure 20); trimethoprim/Sulfamethoxazole (SXT) (figure 21); ciprofloxacin (fig. 22); and ceftriaxone (fig. 23).

FIGS. 24-26 compare chemically sensitive Staphylococcus aureus (S.aureus) to various antimicrobial agentsS. aureus) (of "QC strain 29213") strain. The antimicrobial agent used was vancomycin (figure 24); penicillin (fig. 25); and teicoplanin (fig. 26).

FIGS. 27 and 28 are graphs showing MIC results using the method of the invention directly on clinical samples and compared to clinical results obtained with a Beckman-Coulter MicroScan Walkaway in which the sub-incubation step was performed before overnight growth.

Fig. 29 is a graph showing the detected fluorescence (via a europium containing signaling agent and using wheat germ agglutinin that specifically binds gram positive bacteria) of a gram positive bacteria solution relative to the concentration of bacteria in the solution.

Fig. 30 is a graph showing the detected fluorescence of a gram-negative bacteria solution (via a europium containing signaling agent and using polymyxin B that specifically binds gram-negative bacteria) relative to the concentration of bacteria in the solution.

Fig. 31 is a graph comparing MIC values obtained when an antibody-conjugated europium preparation was used as a signaling agent with MIC values obtained when antibody-horseradish peroxidase (HRP) was used as a signaling agent. SXT on this clinical S.aureus by CLSI overnight method (S. aureus) The MIC of the strain is less than or equal to 0.5 mug/ml.

Fig. 32 is a graph showing the Relative Fluorescence Units (RFU) obtained for specific bacterial concentrations of two europium preparations that are non-specifically bound to the bacterial surface.

Fig. 33 is a graph showing RFU obtained for specific bacterial concentrations for two bacterial species of two europium preparations that non-specifically bind to the bacterial surface.

Fig. 34 and 35 are graphs showing RFU obtained for specific bacterial concentrations of various bacterial species of europium preparations non-specifically bound to the bacterial surface.

Fig. 36A to 36C are graphs showing RFU obtained for specific bacterial concentrations of various bacterial species of europium preparations non-specifically bound to the bacterial surface when various washes comprising glutaraldehyde were used.

FIG. 37 is an E.coli strain for europium preparations that bind nonspecifically to the bacterial surface using a two-step process comprising NH 2-PEG-biotin followed by streptavidin-europium (Eu-SAv) (E.coli) andE. coli) Specific bacterial concentrations obtained.

FIG. 38 is a schematic representation showing the use of a conjugate comprising NHS-LC-LC-biotin E.coli for europium preparations that bind nonspecifically to the bacterial surface followed by a two-step method of Eu-SAv (E.coli)E. coli) Specific bacterial concentrations obtained.

FIG. 39 is a schematic illustrating the confounding effect of a volume-based assay of filamentous growth on antimicrobial sensitivity of a microorganism. Sensitive bacteria that enter filamentous growth may exhibit incorrect resistance due to their increased volume.

Fig. 40 is a schematic diagram illustrating a method for minimizing interference of filamentous microorganisms in the AST assay.

FIG. 41 is a graph showing that E.coli (at concentrations well above MIC) was treated with and without 100. Mu.g/ml ampicillinE. coli) And ampicillin resistant E.coli: (E. coli) Graph of assay results using signaling agents comprising fluorescent nanoparticles.

Fig. 42 is a graph showing the results of assays using signaling agents comprising fluorescent nanoparticles for e.coli with different ampicillin concentrations. Error bars show the standard deviation of triplicates.

FIG. 43 shows Escherichia coli (E. coli) Graph of the ability of functionalized magnetic beads to bind and isolate intact bacteria from solution.

Fig. 44 is a graph showing the number of intact bacteria isolated by functionalized magnetic beads from solutions containing varying amounts of antimicrobial agent.

Figure 45 includes a graph comparing the number of intact bacteria isolated by centrifugation of vs functionalized magnetic beads from solutions containing varying amounts of antimicrobial agent (here vancomycin "VAN"). VAN against this clinical Staphylococcus aureus by CLSI overnight method (S. aureus) MIC of the strain was 8 μ g/ml.

FIGS. 46A to 46C show the design and performance of the tetramino metal organic ligand (TAML) nano markers. Fig. 46A is a schematic diagram showing the composition of a nano-tag. FIG. 46B is a graph (inset) showing catalytic comparison of HRP and TAML; the dashed line is a linear best fit for each data set, where R2 for HRP is 0.997 and R2 for TAML is 0.987. Fig. 46C is a graph showing TAML nanomarker vs HRP comparison for clostridium difficile (c.difficile) toxin a immunoassay. In fig. 46B and 46C, the signal was homogenized to "1" at zero concentration, the propagation error, and the error bars represent ± 1 standard deviation. The experiment was repeated three times in triplicate with similar results.

Definition of

In order that the invention may be more readily understood, certain terms are first defined below. Additional definitions for the following terms and other terms are set forth throughout the specification.

As used in this specification and the appended claims, the singular forms "a", "an" and "the" include plural referents unless the context clearly dictates otherwise.

As used herein, the term "or" is understood to be inclusive and to cover both "or" and "unless specifically stated or apparent from the context.

As used herein, the terms "such as" and "i.e.," are used by way of example only and are not intended to be limiting, and should not be construed to refer only to those items explicitly enumerated in the specification.

The terms "one or more," "at least one," "more than one," and the like are understood to include, but are not limited to

At least 1,2,3, 4,5, 6,7, 8,9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 84, 85, 86, 87, 88, 89, 90, 91 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, or 150, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 2000, 3000, 4000, 5000, or more and any number in between.

Conversely, the term "not more than" includes every value that is less than the recited value. For example, "no more than 100 nucleotides" includes 100, 99, 98, 97, 96, 95,94, 93, 92, 91, 90, 89, 88, 87, 86, 85, 84, 83, 82, 81, 80, 79, 78, 77, 76, 75, 74, 73, 72, 71, 70, 69, 68, 67, 66, 65, 64, 63, 62, 61, 60, 59, 58, 57, 56, 55, 54, 53, 52, 51, 50, 49, 48, 47, 46, 45, 44, 43, 42, 41, 40, 39, 38,37, 36, 35, 34, 33, 32, 31, 30, 29, 28, 27, 26, 25, 24, 23, 22, 21, 20, 19,18, 17, 16, 15, 14, 13, 12, 11, 10, 9,8, 7,6, 5,4, 3,2, 1 and 0 nucleotides.

<xnotran> " ", " ", " ", " " 2,3, 4,5, 6,7, 8,9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149 150, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 2000, 3000, 4000, 5000 . </xnotran>

Throughout this specification the word "comprise", or variations such as "comprises" or "comprising", will be understood to imply the inclusion of a stated element, integer or step, or group of elements, integers or steps, but not the exclusion of any other element, integer or step, or group of elements, integers or steps.

Unless otherwise indicated or apparent from the context, as used herein, the term "about" is understood to be within the normal tolerances in the art, e.g., within 2 standard deviations of the mean. "about" can be understood to be within 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.1%, 0.05%, 0.01%, or 0.001% of the stated value. Unless otherwise clear from the context, all numbers provided herein are modified by the term "about".

The surface may be the outer surface of a cell wall, cell envelope, plasma membrane or cell capsule; the inner surface of a cell wall, cell envelope, plasma membrane or cell capsule; or within a cell wall, cell envelope, plasma membrane or cell capsule. The surface may include cellular structures protruding from the cell including, but not limited to, cilia, pili, and flagella. The surface may comprise an organelle. The surface may include transmembrane proteins, cell wall proteins, extracellular proteins, intracellular proteins, extracellular associated polysaccharides, intracellular associated polysaccharides, extracellular lipids, intracellular lipids, membrane lipids, cell wall lipids, proteins, polysaccharides, and/or lipids integrated with or associated with the cell envelope. The surface may comprise nucleic acids.

The surface may comprise biomolecules bound or associated with a signaling agent. Exemplary biomolecules include peptidoglycans, mureins, mannoproteins, porins, beta-glucans, chitins, glycoproteins, polysaccharides, lipopolysaccharides, lipooligosaccharides, lipoproteins, endotoxins, lipoteichoic acids, teichoic acids, lipid a, carbohydrate binding domains, efflux pumps, other cell wall and/or cell membrane associated proteins, other anionic phospholipids, and combinations thereof.

Growth, such as in the growth of microorganisms, includes proliferation of the number of microorganisms, an increase in length, an increase in volume, and/or an increase in nucleic acid and/or protein content.

The control may include an antimicrobial agent to which the microorganism is not susceptible. For example, if the assay is used to determine the sensitivity of gram-positive bacteria, the control (and test incubates) may include one or more antimicrobial agents that target gram-negative bacteria, and if the assay is used to determine the sensitivity of eukaryotic microorganisms, the control (and test incubates) may include one or more antibacterial antimicrobial agents.

The control may be a positive control measured from the microorganism under otherwise identical conditions, but without the antimicrobial agent or with the antimicrobial agent to which one or more microorganisms are not susceptible.

The control can be measured from the microorganism under otherwise identical, but nutrient-free conditions.

The control can be measured from the microorganism under otherwise identical conditions with one or more toxins known to inhibit the growth of the microorganism.

The control may be a historical control. Here, the test incubation may be performed after the control incubation has been performed.

Alternatively, the control may be performed in a different cartridge than the cartridge containing the test incubations.

By "treated" is meant a step of isolating a microorganism from a biological sample, a step of increasing the concentration of a microorganism obtained from a biological sample, and/or a step of increasing the number of microorganisms obtained from a biological sample, for example by culturing the microorganisms under conditions that promote proliferation of the microorganisms.

The compounds of the present invention include those generally described herein and are further illustrated by the classes, subclasses, and species disclosed herein. As used herein, the following definitions shall apply unless otherwise indicated. For the purposes of the present invention, chemical elements are identified according to the periodic Table of the elements, CAS edition, handbook of Chemistry and Physics, 75 th edition. Furthermore, the general principles of Organic Chemistry are described in "Organic Chemistry", thomas Sorrell, university Science Books, sausaltito: 1999 and "March's Advanced Organic Chemistry", 5 th edition, editions Smith, M.B., and March, J., john Wiley & Sons, new York: 2001, the entire contents of which are incorporated herein by reference.

The term "heterocyclic", "heterocyclyl" or "heterocyclic" as used herein means a non-aromatic, mono-, bi-or tricyclic ring system in which one or more ring members are independently selected heteroatoms. In some embodiments, a "heterocyclic", "heterocyclyl", or "heterocyclic" group has three to fourteen ring members in which one or more ring members are heteroatoms independently selected from oxygen, sulfur, nitrogen, or phosphorus, and each ring in the system contains 3 to 7 ring members.

The term "heteroatom" refers to one or more of oxygen, sulfur, nitrogen, phosphorus, and silicon (including any oxidized form of nitrogen, sulfur, phosphorus, or silicon; quaternized form of any basic nitrogen or; heterocyclic substitutable nitrogen, e.g., N (as in 3,4-dihydro-2H-pyrrolyl), NH (as in pyrrolidinyl), or NR + (as in N-substituted pyrrolidinyl)).

The term "alkoxy" or "thioalkyl" (thioalkyl) as used herein refers to an alkyl group, as previously defined, which is attached through an oxygen ("alkoxy") or sulfur ("thioalkyl") atom.

The terms "haloalkyl", "haloalkenyl", "haloalkoxy" and "haloalkoxy" mean alkyl, alkenyl or alkoxy, as the case may be, substituted with one or more halogen atoms. The term includes perfluorinated alkyl groups such as-CF 3 and-CF 2CF3.

The terms "halogen", "halo" and "halo" mean F, cl, br or I.

The terms "aryl" and "ar-" used alone or as part of a larger moiety (e.g., "aralkyl", "aralkoxy", or "aryloxyalkyl") refer to an optionally substituted C6-14 aromatic hydrocarbon moiety containing one to three aromatic rings. For example, aryl groups are C6-10 aryl groups (i.e., phenyl and naphthyl). Aryl groups include, but are not limited to, optionally substituted phenyl, naphthyl, or anthracenyl. The terms "aryl" and "ar-" as used herein also include groups in which an aryl ring is fused to one or more cycloaliphatic rings to form an optionally substituted cyclic structure such as a tetrahydronaphthyl, indenyl, or indanyl ring. The term "aryl" may be used interchangeably with the terms "aryl group", "aryl ring" and "aromatic ring".

The compounds of the invention may be present in free form for use in therapy or, where appropriate, as pharmaceutically acceptable salts.

As used herein, the term "aromatic" includes aryl and heteroaryl groups as generally described below and herein.

The term "aliphatic" or "aliphatic group" as used herein means an optionally substituted straight or branched chain C1-12 hydrocarbon that is fully saturated or which contains one or more units of unsaturation. For example, suitable aliphatic groups include optionally substituted straight or branched chain alkyl, alkenyl, and alkynyl groups. Unless otherwise specified, in various embodiments, aliphatic groups have 1 to 12, 1 to 10, 1 to 8,1 to 6,1 to 4, 1 to 3, or 1 to 2 carbon atoms. It will be apparent to those skilled in the art that in some embodiments, the "aliphatic" groups described herein may be divalent.

The term "alkyl", used alone or as part of a larger moiety, refers to a saturated, optionally substituted, straight or branched chain hydrocarbon radical having 1-12, 1-10, 1-8, 1-6, 1-4, 1-3, or 1-2 carbon atoms.

The term "alkenyl" used alone or as part of a larger moiety refers to an optionally substituted straight or branched chain hydrocarbon group having at least one double bond and having 2-12, 2-10, 2-8, 2-6, 2-4, or 2-3 carbon atoms.

The term "alkynyl", used alone or as part of a larger moiety, refers to an optionally substituted straight or branched chain hydrocarbon group having at least one triple bond and having 2-12, 2-10, 2-8, 2-6, 2-4 or 2-3 carbon atoms.

Unless otherwise indicated, the structures depicted herein are also intended to include all isomeric (e.g., enantiomeric, diastereomeric, and geometric (or conformational)) forms of the structure; for example, the R and S configurations of each asymmetric center, (Z) and (E) double bond isomers, and (Z) and (E) conformational isomers. Thus, single stereochemical isomers as well as enantiomeric, diastereomeric, and geometric (or conformational) mixtures of the compounds of the invention are within the scope of the invention. Unless otherwise indicated, all tautomeric forms of the compounds of the invention are within the scope of the invention. Furthermore, unless otherwise indicated, the structures depicted herein are also intended to include compounds that differ only in the presence of one or more isotopically enriched atoms. For example, compounds having the present structure wherein hydrogen is replaced by deuterium or tritium, or carbon is replaced by 13C-or 14C-enriched carbon are within the scope of the present invention. By way of non-limiting example, such compounds may be used as analytical tools or probes in bioassays.

It is to be understood that when the disclosed compounds have at least one chiral center, the invention encompasses an enantiomer of the inhibitor free of the corresponding optical isomer, racemic mixtures of the inhibitor and mixtures enriched in one enantiomer relative to its corresponding optical isomer. When a mixture is enriched in one enantiomer relative to its optical isomers, the mixture contains, for example, an enantiomeric excess of at least 50%, 75%, 90%, 95%, 99%, or 99.5%.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs and as commonly used in the art to which this application belongs, and this field (art) is incorporated by reference in its entirety. In case of conflict, the present specification, including definitions, will control.

Detailed Description

The invention allows for the rapid determination of antibiotic susceptibility of microbial infections. The present invention is based, in part, on the surprising discovery of non-specific surface binding assays that provide accurate and rapid Antimicrobial Sensitivity Test (AST) assays in less than twelve hours-and, in particular, in 4 hours. The present invention ("fast-AST") provides accurate results consistent with those obtained using the Clinical Laboratory Standards Institute (CLSI) reference method and when tested with a variety of antimicrobial agents and on a variety of microorganisms; however, the present invention takes significantly less time to obtain results compared to the CLSI method. Furthermore, the present invention accurately distinguishes between an antimicrobial MIC of a clinically relevant strain of a microorganism that is resistant to one or more antimicrobial agents and an antimicrobial MIC of a strain of the same microorganism that is susceptible to the antimicrobial agent. Furthermore, the invention may include signaling agents (e.g., europium compounds) that bind non-specifically, rather than specifically, to the microorganism (e.g., via chemically conserved groups or biochemically conserved binding sites on the microorganism), thereby extending the generality of the invention to any microorganism and allowing the appropriate treatment to be initiated without first identifying a particular infectious microorganism. Furthermore, the invention allows signal amplification, such that microorganisms can be detected rapidly at lower concentrations, e.g. from diluted cultures of microorganisms or via biological samples of patients. Furthermore, the present invention can use europium formulations as the chemical moiety, thereby extending the dynamic range of the method and allowing more accurate assays to be performed from a range of microbial samples. Finally, the present invention is compatible with existing equipment, thereby enabling rapid adoption in current clinical laboratories. Thus, the present invention can provide patients with an appropriate treatment regimen, i.e., a specific antimicrobial agent and at a specific dosage, in a substantially reduced amount of time and expense relative to standard methods. Thus, the present invention will improve patient outcomes, reduce hospital costs, and help reduce the further evolution of antimicrobial resistant microorganisms; the present invention therefore represents a significant breakthrough in the AST field.

Aspects of the invention provide accurate, low cost phenotypic AST results by chemically amplifying the microbial surface. This novel process offers two major advances over the currently used processes: 1) Quantifying microbial growth by determining relative surface area, which overcomes the current platform limitations with respect to filamentous growth regimes, as is well known to those skilled in the art; and 2) 1 × 10 using standard optical detection equipment ³ To 1 × 10 ⁸ Optimal sensitivity of microbial amplification in the CFU/ml range.

As disclosed herein (e.g., in the examples), the present invention has been shown to work for a wide range of microbial species, including all six (enterococcus faecium: (enterococcus faecium)) (ii)Enterococcus faecium) Staphylococcus aureus (1)Staphylococcus aureus) Klebsiella pneumoniae (K.pneumoniae) ((B))Klebsiella pneumoniae) Bob's disease Acinetobacter (A), (B), (C)Acinetobacter baumannii) Pseudomonas aeruginosaPseudomonas aeruginosa) And enterobacteriaceae species (Enterobacterspecies) ("ESKAPE") pathogen provided equivalent results to gold standards. Due to the generality of the present invention it is flexible as it can be easily and cheaply adapted to new microbial species strains and diagnostic tests.

The present invention provides a low cost phenotypic AST from a standard microbial colony isolate or directly from a positive blood sample in less than 8 hours, preferably less than 5 hours. This allowed standard clinical microbiology laboratories to move with, phenotypic AST results. The following working examples show that chemical amplification of a microbial surface produces accurate Minimum Inhibitory Concentrations (MIC) and breakpoint calls (breakpoint calls) in less than four hours. This will shorten the current latency by more than 20 hours and will match the current MALDI-TOF identification from positive blood cultures that is close to FDA testing and the multiplex PCR identification platform from positive blood cultures that has obtained FDA approval. This design enables the present invention ("fast-AST" platform) to break the traditional speed vs. cost tradeoff. The present invention is compatible with both standard microplate formats (e.g., having 6, 12, 24, 48, 96, 384, or 1536 wells) and conventional optical detectors.

The identification of invading pathogens and Antimicrobial Susceptibility Testing (AST) with speed and accuracy allows for the timely administration of the most effective therapeutic agents. This treatment ameliorates infection, reduces hospitalization of hospitalized patients, and shortens the time that patients are subjected to a broad spectrum antimicrobial, which contributes to the global prevalence of antimicrobial resistance. In contrast, the currently accepted wait of more than 30 hours for microbiological identification and sensitivity results requires overuse of broad spectrum antimicrobials and longer patient stays than necessary. For this reason, the counseling board for general systems to combat antibiotic-resistant bacteria has recently made the development and use of rapid diagnostics for the detection of antibiotic-resistant bacteria one of its primary goals.

The present invention, which results in a faster and accurate AST determination, can provide a practical cost benefit of over $ 2,000 per patient. These points of value include easier quantifiability (reduced length of residence time and expensive treatment) and more intangible, difficult to assess (patient mortality and social impact from improved antimicrobial management efforts). Some of these intangible values, such as the value of antimicrobial management work, can become more quantitative as regulatory agencies are beginning to impose costs on hospitals as they do not adopt stricter antimicrobial management work plans. In 9 months 2014, the academy of participating, california act 1311 was signed as a law, further requiring hospitals to adopt and enforce antimicrobial management policies according to guidelines established by federal government and professional organizations, and establishing multidisciplinary antimicrobial management committees with physician supervision by at least one physician or pharmacist who has undergone specific training in connection with management. In 2016, the center for the health care and health care subsidy system (CMS) used a suggested role to facilitate antimicrobial management in hospitals, where many industry experts are expected to implement financial incentives within the next two years. The present invention will further drive the goal of better antimicrobial management by the government and the healthcare industry.

The general steps of aspects of the present invention are shown in FIG. 1. The image in FIG. 1 shows aspects with different processing steps; however, aspects of the invention may be automated.

Fig. 2A to 2D show features of aspects of the present invention. FIG. 2A shows the sensitivity range for detection of three representative pathogens. The dashed line shows the zero concentration signal level. FIG. 2B shows a "Crocodile" (Titertek-Berthold) automated Rapid-AST prototype platform that can be used in the present invention. Figure 2C is a schematic showing anionic bacteria interacting with cationic nano-tags and polymers. The resulting reduced solubility of the neutral complex allows the magnetic beads to bind. FIG. 2D shows data for Staphylococcus aureus having the SensiTitre gram positive group (GPALL 3F) which shows bacteriostatic (clindamycin) and bactericidal (penicillin) antimicrobial results relative to the high growth and "freeze time" (FIT) control.

As known to those skilled in the art, the AST platform may produce Minimum Inhibitory Concentration (MIC) results and/or Qualitative Sensitivity Results (QSR) for each antimicrobial agent tested. MIC is generally known to be the lowest concentration of antimicrobial that inhibits microbial growth and provides dosing information to physicians. QSR may also provide similar dosage information to physicians, but may not provide a digital MIC. The AST assay is primarily configured to test multiple antimicrobial agents in parallel for each obtained biological sample. To generate MIC or QSR results, a dilution series is required for each antimicrobial agent. Thus, for CLSI liquid-based AST, referred to as "liquid medium microdilution", assays are typically performed in cartridges and/or microplates, which enable parallel testing of different antimicrobial agents at different concentrations.

The long time to obtain AST measurements results in incomplete information being passed to the physician. These long periods of time often prevent the rate or kinetics of killing from being identified for the effectiveness of the antimicrobial agent. This additional information may be important to inform the treatment. Current AST, which is not determined until more than 6 hours (and often more than 12 hours) after treatment initiation, often loses the ability to discern a difference between the rates of antimicrobial efficacy: the antimicrobial agent that kills the microorganism immediately after 12 hours appears the same as the antimicrobial agent that kills it within four hours.

Table 1 estimates the effect of different treatments on bacterial numbers after 2 hours incubation. Assuming a doubling time of 30 minutes, the untreated control should increase by a factor of 16. A treatment group with an "effective" antimicrobial agent (defined as an antimicrobial agent that is effective against, e.g., bacteria) above the MIC should result in minimal microbial growth and, in the case of a bactericidal antimicrobial agent, microbial death. Thus, less bacteria than the starting concentration is expected. A treatment group with an "effective" antimicrobial agent below its MIC should result in microbial growth equal to or less than a control without the antimicrobial agent. Slow acting antimicrobials, defined in this case as those that require more than two hours to kill bacteria (e.g. as is the case with bacteriostatic antimicrobials), will produce a signal between the initial concentration and a 16 fold increase.

TABLE 1

Step (ii) of	No antimicrobial agent	Effective antimicrobial agents at the following concentrations	Step (ii) of	No antimicrobial agent
					Initial bacterial concentration	5×10 5	5×10 5	5×10 5	5×10 5
Estimated bacterial concentration after 2 hours, with a doubling time of 30 minutes	8×10 6	≤8×10 6	<5×10 5	5×10 5 To<8×10 6

For the liquid medium microdilution technique, 5X 10 ⁵ The starting concentration of bacteria in CFU/ml is given in the American society for microbiology in 2005 "Manual of Antimicrobial Suadaptability Testing" (where Marie B. Coyle is a harmonized editor). Since each well contains approximately 100 μ L, there is approximately 5X 10 per well ⁴ And (4) bacteria. The standard fluorescent dye starts at approximately 0.1 nM concentration (which corresponds to approximately 1.2 × 10) ¹⁰ Individual molecules) can be quantified. Thus, for a 30 minute doubling time bacterium to be visible after two hours, 1.5X 10 must be used for each individual bacterium ⁴ And (4) labeling the fluorescent molecules. Practical considerations, such as fluorescent background and non-specific binding, can increase this number by orders of magnitude. In order to enable compatibility with standard optical detectors, it may therefore be advantageous to use chemical and/or biochemical amplifiers capable of producing detectable signals at lower concentrations.

Without wishing to be bound by theory, the present invention is based in part on the principle of microdilution of liquid media. The culture to be evaluated is diluted, most preferably to 1-10X 10 ⁵ CFU/ml and introduced into wells containing different antimicrobial agents at different concentrations, so that the MIC of the appropriate set of antimicrobial agents can be determined. The plate is then introduced into an incubator at an appropriate temperature (most preferably 31-37 ℃) and under appropriate conditions (most preferably aerobic) for bacterial growth. During this time, the microorganisms can grow。

The liquid medium may be a cation-conditioned Mueller Hinton liquid medium and may contain additional supplements known to those skilled in the art to facilitate microbial growth (e.g., lysed horse blood) and/or for determining antimicrobial efficacy (e.g., high sodium chloride concentration). The microplate may be shaken during this growth, which may facilitate dispersion of nutrients and/or gas exchange and/or antimicrobial agents in each well and/or reduce biofilm formation.

A known amount of signaling agent is added to each well within 0 to 8 hours (most preferably 0 to 4 hours) of the AST start. The addition of reagents (including signal generating agents) may be performed by automated or semi-automated instruments, or may be performed manually.

The signaling agents (which may be referred to as "viscosity-amps") comprise a moiety capable of binding to a microorganism (e.g., an antibody and/or lectin bound to the surface of the microorganism, a charged moiety and/or functional moiety that non-specifically binds to the surface of the microorganism) and a chemical moiety capable of providing a signal or contributing to the generation of a signal (e.g., enzymatic chemiluminescers and lanthanide chelates). Exemplary enzymes include horseradish peroxidase, alkaline phosphatase, acetylcholinesterase, glucose oxidase, beta-D-galactosidase, beta-lactamase, and combinations thereof.

As used herein, a signal-generating agent may include one or more chemical moieties (i.e., "signal-generating agents") conjugated to one or more "microbial receptors. Signal-generating agents include, but are not limited to, one or more catalysts (including enzymes, metal oxide nanoparticles, organometallic catalysts, nanoparticles designed for signal amplification (such as those described in U.S. provisional application, to which priority is claimed herein and incorporated by reference in its entirety), bacteriophages containing signal-generating elements, fluorophores (including organic fluorophores, europium or ruthenium (II), rhenium (I), palladium (II), platinum (II) -containing organometallic compounds), and/or colorimetric dyes (including organic "dyes"). Combinations of the foregoing can be used, such as nanoparticles, dendrimers, and/or other nanoscale structures having enzymes, fluorophores, and/or organometallic molecules.

The chemical moiety may be conjugated to the signaling agent either simultaneously with the initial contact of the signaling agent with the microorganism prior to contacting the signaling agent with the microorganism, or after the signaling agent has contacted the microorganism.

When the signaling agent is added to the AST dilution containing the microorganism, a signaling agent receptor (e.g., a moiety that can specifically or non-specifically bind the microorganism) is associated with the surface of the microorganism. Thus, the more intact the microorganism, e.g., in solution, the greater the amount of signaling agent that will be associated with these bacteria. Thus, there is an inverse relationship between the number of intact bacteria and the number of "free" signaling agents in solution (as defined by cells not bound to intact bacteria). Note that if, for example, a microorganism is lysed in response to treatment with an antimicrobial agent, the free signaling agent may bind to the soluble microbial component.

The amount of signaling agent associated with and/or inserted into the surface of a microorganism is directly proportional to the surface area of the microorganism. The surface area of the microorganisms is closely related to the actual resistant microorganisms. In particular, in the case of microorganisms (e.g., filament-forming bacteria) that swell or elongate in response to MIC and sub-MIC concentrations of antimicrobial agents, metabolic and/or volumetric identification is known to give a pseudo-sensitivity profile for "rapid" AST time points (defined as those less than 6 hours). To overcome this limitation, the present invention converts the surface area (rather than volume) of the microorganism into a measurable signal, most preferably an optical signal. The method enables accurate determination of a microbial resistance profile in less than 6 hours.

In order to separate the signaling agent associated with and/or inserted into the microorganism from the free signaling agent, it is necessary to perform one or more separation and/or competitive binding steps. Such steps include, but are not limited to, centrifugation (e.g., tog-force>500 × g) Filtration (e.g., via a filter having pores less than or equal to 0.45 microns, and preferably less than or equal to 0.2 microns), electrophoretic and/or magnetic capture; such steps are well known to those skilled in the art.

To facilitate the binding of the signaling agent and/or to reduce the background, it may be further advantageous to separate the microorganism from the liquid in which it is suspended during incubation before the addition of the signaling agent. Such separation may include, but is not limited to, centrifugation, filtration, electrophoresis, and/or magnetic capture.

When these data are compared between treatment groups, a microbial resistance profile can be determined using procedures similar to the AST assay currently used. In addition, these data may enable the determination of the rate of antimicrobial efficacy or killing kinetics.

The signaling agents may be added with the microorganisms and/or antimicrobial agents such that they are present throughout the AST incubation period. The total period of time may be up to twenty-four hours, but is preferably within eight hours, and more preferably within five hours. Alternatively, the signaling agent may be added to the microorganism and antimicrobial agent after a defined incubation period. The period of time may be up to twenty four hours, but is preferably within eight hours, and more preferably within four hours.

The signaling agent is designed to associate with and/or embed a microbial surface (including a wall and/or a membrane). Signaling agents designed for association comprise a binding moiety, including but not limited to one or more antibodies, lectins, other proteins, small molecules with one or more charged chemical groups, small molecules with one or more functional chemical groups, bacteriophages, glycoproteins, peptides, aptamers, charged small molecules, small molecules with fixed charge, charged polymers with fixed charge, hydrophobic small molecules, charged peptides with fixed charge, peptides with alternating hydrophilic and hydrophobic regions, and/or small molecule ligands, which may or may not be organometallic complexes. The design of molecules for microbial association is well known to those skilled in the art. The signaling agent may remain bound to the microbe and/or internalized, thus including all associations. Signaling agents designed for insertion may include, but are not limited to, small hydrophobic molecules, hydrophobic peptides, and/or peptides having alternating hydrophobic and hydrophilic regions. The design of molecules for microbial insertion is well known to those skilled in the art. The signaling agent may further be specific for one or more types of microorganisms. The signaling agent may have multiple receptors. These may enhance binding and/or enable simultaneous binding of two or more microorganisms, which may further be used to "agglutinate" the bacteria. It may be advantageous to adjust the pH of the solution prior to or simultaneously with the addition of the signaling agent. This may be advantageous to enhance the charge-charge interaction between the microorganism and the signaling agent. By titrating the solution pH above neutral (more basic), the anionic charge of the microorganisms can be increased. Thus, it may be advantageous to use moieties having one or more fixed cationic charges.

Notably, the signaling agent can specifically bind to the microorganism (e.g., an antibody that specifically binds to a microorganism species or strain of microorganism) or can non-specifically bind to the microorganism (e.g., through universal covalent or non-covalent bond formation and another non-specific chemical association known in the art).

Preferably, the signaling agent binds to a natural microbial surface.

Alternatively, a chemical and/or biochemical capable of associating with a signaling agent may be added to the liquid in which the microorganism is suspended during growth, such that the chemical and/or biochemical is incorporated into the microorganism during incubation. This may be used to enhance the association of the signaling agent with the microorganism. In alternative embodiments, the signaling agent itself may be present in the liquid in which the microorganism is suspended during incubation, and may be incorporated into the microorganism during growth.

Preferably, the signalling agent comprises an amplifier signal generating agent, such that the signal from each intact microorganism can be amplified beyond the number of signalling agents associated with each microorganism. For example, the enzyme horseradish peroxidase (HRP) is known to amplify signals>1×10 ⁴ And (4) multiplying. Thus, if 100 HRP molecules are bound to each microbial surface, 10 can be achieved ⁶ Amplification of (3). This may increase AST assays performed by enabling discrimination of microorganism concentrations that would otherwise be indistinguishableSpeed. The use of europium formulations similarly provides signal amplification.

Alternatively, the signaling agent may comprise an optical dye precursor known to those skilled in the art as a "membrane dye" designed to greatly increase fluorescence emission upon insertion into a hydrophobic region (such as a cell membrane). Assays designed with these signaling agents may require concentrating the microorganisms to a smaller volume, near planar, to produce sufficient signal to easily perform optical measurements. Interfering substances may require the use of near-IR fluorophores.

Potential separation techniques include, but are not limited to, filtration (e.g., via a filter having pores less than or equal to 0.45 microns, preferably less than or equal to 0.2 microns), centrifugation (e.g., tog-force>500 × g) Electrophoresis, dielectrophoresis and magnetic trapping. These techniques are used to separate signaling agents associated with microorganisms from those free in solution, which microorganisms are attached to filters, precipitated in centrifuges, and/or electrophoretically and/or magnetically separated. Free signaling agents pass through a filter ("filtrate"), remain in solution ("supernatant") after centrifugation or magnetic separation, and/or are run electrophoretically separately. Centrifugation may be standard, density gradient or differential centrifugation. Magnetic separation may require the addition of one or more magnetic particles that specifically target the association or binding with the microorganism. These may be added before or simultaneously with the addition of the signalling agent.

Such separation techniques can also separate microorganisms that change morphology in response to antimicrobial treatment, and can confound assays. One example of such a microorganism is a filamentous bacterium, which initially elongates in response to an antimicrobial treatment. Such growth modes are known to those skilled in the art. The isolation and exclusion of filamentous bacteria from an assay using the isolation techniques described herein will increase the accuracy of the results obtained.

Microbial isolation may be enhanced by association of the particles with microbial species. For example, in the case of magnetic separation, magnetic beads may be associated with a microorganism (specifically or non-specifically). The moiety present on the surface of the magnetic bead may be the same surface as the signal transduction agent to which the microorganism (or a biological organism thereof) is boundMolecules) or different surfaces (or biomolecules thereof). The magnetic beads can have the same and/or different moieties as the signaling agent. For example, if the signaling agent comprises a conjugated E.coli: (A.coli) ((B.coli))E. coli) The magnetic beads may then be functionalized with the same antibody. In other examples, the signaling agent can include a motif that binds the microorganism, and the magnetic beads are functionalized to non-specifically bind the microorganism.

The one or more binding moieties associated with the magnetic beads can be the same as or different from the chemical moiety associated with the signaling agent or the chemical moiety of the signaling agent.

The one or more binding moieties associated with the magnetic beads can bind the microorganism before, simultaneously with, or after the signaling agent binds to the microorganism.

One or more binding moieties associated with the magnetic beads can be associated with one or more polymers that precipitate the microorganisms. The one or more polymers that precipitate the microorganisms can be cationic. The one or more microorganism-precipitating polymers may be poly (ethylene glycol).

The magnetic beads may be in the size range of 20nm to 20 microns as known to those skilled in the art.

After isolation, one or more assays may be performed to determine the amount of signaling agent remaining after isolation of the microorganism and/or the amount of signaling agent removed during isolation of the microorganism ("free" signaling agent). Performing an assay for the free signaling agent provides a signal that is inversely proportional to the concentration of the microorganism. In this case, the signaling agent associated with the microorganism may be associated with or internalized by the microorganism. Alternatively, the assay can be performed for a signaling agent associated with the microorganism. In this case, unless the microorganism is specifically lysed, only the bound signaling agent will contribute to the signal.

To maximize the efficiency of the separation, even if the amount of free signaling agent remaining is minimized, one or more washing steps may be performed. These may be continuous, as in the case of filtration, magnetic capture or electrophoresis, and/or discrete, as in the case of centrifugation or magnetic capture.

In alternative embodiments, the signaling agent may not require washing. This may be the case when a "membrane dye" signaling agent is used. Molecules that are not inserted into the microbial membrane have significantly lower optical activity than the inserted material and therefore may not require washing.

One or more washes may be performed prior to adding the signaling agent to the microorganism. For example, these washes can remove interfering substances present in the liquid in which the microorganisms are suspended during incubation.

In embodiments, no washing is performed.

Signal development may require the addition of a "developing solution". For signaling agents comprising a catalyst, the chromogenic solution may comprise one or more signal precursors, which may be converted to optically and/or electrically active signaling molecules. For signaling agents comprising encapsulated molecules (such as within nanoparticles), the chromogenic solution may comprise one or more agents to release the encapsulated substance. At a specified time after addition of the chromogenic solution, colorimetric and/or electrochemical signals may be measured. Such signals include, but are not limited to, absorbance, fluorescence, time-resolved fluorescence, chemiluminescence, electrochemiluminescence, amperometry, voltammetry, impedance, and/or impedance spectroscopy. The data can then be compared to determine the ASTs and MICs, which are similar to current AST protocols.

In embodiments, determining the signal level comprises measuring the signal level associated with the intact microorganism. Alternatively or additionally, determining the signal level comprises measuring a signal level that is not associated with an intact microorganism.

These processes can be performed directly from cultures, sub-cultures, positive blood cultures, samples. Treatment to concentrate microorganisms and/or remove potentially interfering substances may be performed prior to AST or prior to addition of signaling agents.

Signaling agents may also be used with plate-based methods for AST assays, such as gradient diffusion. They can be added simultaneously after the addition of the microorganisms to the plate or after a set incubation period. In these cases, spatial information of the optical and/or electrical signals is important. With this method, the determination of the signaling agent bound for the intact microorganism may be preferred in order to preserve spatial information. In this case, one or more washing steps may be performed prior to addition of the chromogenic solution in order to remove free signaling agents.

In embodiments, no washing is performed.

Alternatively, the signaling agent may be designed to be taken up by the bacteria, which may be achieved, for example, through the use of a bacteriophage. In such methods, an assay for free signaling agent is performed.

Alternatively, a blot transfer method, such as that standard with nitrocellulose paper, can be used to transfer bacteria or free signaling agents, and then a spatial assay is performed on the blot paper.

If the signaling agent produces a signal upon binding, the separation step(s) may not be required. Alternatively, if the signaling agent becomes sensitive or resistant to a particular developer solution component upon binding, a process without a separation step can be achieved.

The final MIC and/or QSR output data may be interpreted by the user directly from the data generated by the assays described herein. Alternatively, the data may be processed by one or more algorithms to derive MICs and/or QSRs. The reported MIC and/or QSR values may be derived from one or more assays described herein, or may be derived from one or more assays described herein in conjunction with one or more known assays for microbial growth, including but not limited to metabolic dye indicator assays, pH indicator assays, nucleic acid assays, and ATP assays.

Method of the invention

One aspect of the invention is a method for determining antimicrobial sensitivity of a microorganism. The method comprises the following steps: incubating a liquid suspension of a microorganism in the presence of an antimicrobial agent and a signaling agent under conditions that promote growth of the microorganism, wherein the signaling agent is capable of binding to the surface of the microorganism; separating microorganisms bound by the signaling agent from unbound signaling agent; and determining the level of signal associated with the microorganism as compared to one or more controls, thereby determining the antimicrobial sensitivity of the microorganism.

Another aspect of the invention is a method for determining antimicrobial sensitivity of a microorganism. The method comprises the following steps: incubating a liquid suspension of microorganisms in the presence of an antimicrobial agent under conditions that promote growth of the microorganisms; adding a signaling agent capable of binding to the surface of the microorganism; separating microorganisms bound by the signaling agent from unbound signaling agent; and determining the level of signal associated with the microorganism as compared to one or more controls, thereby determining the antimicrobial sensitivity of the microorganism. In embodiments, the addition of the signaling agent occurs before or during the incubating step, or the addition of the signaling agent occurs after the incubating step.

Another aspect of the invention is a method for determining antimicrobial sensitivity of a microorganism. The method comprises the following steps: incubating a liquid suspension of microorganisms in a cartridge comprising a plurality of chambers, each chamber containing one or more antimicrobial agents, under conditions that promote growth of the microorganisms; adding a signaling agent to the plurality of chambers, wherein the signaling agent is capable of binding to a surface of a microorganism; removing unbound signaling agent; and determining the level of signaling in the plurality of chambers as compared to the one or more controls, thereby determining the sensitivity of the microorganism to the one or more antimicrobial agents. In embodiments, the cartridge further comprises one or more control chambers (e.g., at least 2,4, 6,8, 12, 24, 48, 96, 192, 384, 1536 or more chambers) that do not contain an antimicrobial agent or an antimicrobial agent to which one or more microorganisms are not sensitive.

In embodiments of the above aspect, the binding to the surface of the microorganism is non-specific, e.g., comprising non-covalent interactions and via formation of covalent bonds.

In embodiments of the above aspects, the signaling agent may comprise a chemical and/or biochemical group capable of binding to a surface of a microorganism, wherein the surface comprises one or more of a membrane, a wall, a protein, an organelle, a sugar, a lipid, a cell envelope, and/or a nucleic acid.

In embodiments of the above aspects, the signaling agent may comprise a chemical and/or biochemical group of a biomolecule capable of binding to the surface of the microorganism, wherein the surface biomolecule is selected from the group consisting of peptidoglycans, mureins, mannoproteins, porins, beta-glucans, chitins, glycoproteins, polysaccharides, lipopolysaccharides, lipooligosaccharides, lipoproteins, endotoxins, lipoteichoic acids, teichoic acids, lipid a, carbohydrate binding domains, efflux pumps, other cell wall and/or cell membrane associated proteins, other anionic phospholipids, and combinations thereof.

In embodiments of the above aspect, the signaling agent may comprise a signal amplifier and one or more chemical moieties capable of non-specifically binding to the surface of the microorganism.

Another aspect of the invention is a method for determining antimicrobial sensitivity of a microorganism. The method comprises incubating the microorganism in the presence of an antimicrobial agent and a signaling agent under conditions that promote growth of the microorganism, wherein the signaling agent comprises a signal amplifier and one or more chemical moieties capable of non-specifically binding to the surface of the microorganism; separating microorganisms bound by the signaling agent from unbound signaling agent; and determining the level of signal associated with the microorganism as compared to one or more controls, thereby determining the antimicrobial sensitivity of the microorganism.

Another aspect of the invention is a method for determining antimicrobial sensitivity of a microorganism. The method comprises incubating the microorganism in the presence of an antimicrobial agent under conditions that promote growth of the microorganism; adding a signaling agent comprising a signal amplifier and one or more chemical moieties capable of non-specifically binding to the surface of the microorganism; separating microorganisms bound by the signaling agent from unbound signaling agent; and determining the level of signal associated with the microorganism as compared to one or more controls, thereby determining the antimicrobial sensitivity of the microorganism. In embodiments, the signaling agent occurs before, at the beginning of, or during the incubating step, preferably during the incubating step. In embodiments, the microorganism is incubated in a liquid suspension.

In embodiments of the above aspect, the liquid suspension may be prepared by inoculating a liquid culture medium with a microbial isolate grown from a biological sample.

In embodiments of the above aspect, the liquid suspension of the microorganism can be prepared from an untreated biological sample, e.g., which has not been subjected to a culturing step.

In embodiments of the above aspects, a liquid suspension of the microorganism can be prepared from the cultured or treated biological sample.

In embodiments of the above aspect, the biological sample is selected from the group consisting of blood, cerebrospinal fluid, urine, feces, vaginal fluid, sputum, bronchoalveolar lavage, throat, nasal/wound swab, and combinations thereof.

In embodiments of the above aspect, the method does not involve the step of capturing the microorganism on a solid surface before or during incubation.

In embodiments of the above aspect, the method does not comprise a step of growing the microorganism on a solid surface during or after the incubating step.

In embodiments of the above aspect, the incubating may comprise shaking the liquid suspension of the microorganism.

In embodiments of the above aspect, the liquid suspension of the microorganism may be agitated continuously or discretely during incubation by means of mechanical, acoustic and/or magnetic stirring.

In an embodiment of the above aspect, the incubation occurs at 31-37 ℃.

Comparison of the present invention with the AST System currently in use

The present invention is advantageous over currently used AST methods in part because it provides accurate AST results in significantly less time.

Three automated AST systems currently used in clinics are Vitek2 from BioMeri ux, phoenix from Beckman Dickinson and MicroScan from Beckman-Coulter. A comparison between the currently used AST system and the steps in the present invention is shown in fig. 3. The process described in the present invention can be performed in at least two modes. The first was used for standard isolates, where the current laboratory workflow was unchanged. The second was directly from positive blood cultures.

For standard isolate processing, the present invention is compatible with existing clinical laboratory workflows and therefore does not require changes. As shown in "step 8" of fig. 3, the current sensitivity (AST) test is performed after colony isolation (step 6) and normalization of microorganism concentration (step 7). In this workflow, the present invention will replace the current system at "step 8". Because the AST results of the present invention are available within the transition (< 5 hours) of the healthcare worker, in practice, the utility may increase the speed at which the patient receives the optimized therapy (step 9) by up to one day. The automation may be designed to include "step 7" and potentially additional steps in the workflow. Such automation is known to those skilled in the art. Note that fig. 3 illustrates the workflow of a blood sample. Many sample types, such as urine and swabs, can be directly streaked onto the plate (step 4). In this case, gram staining may be performed at step 6.

For blood testing, the AST systems currently used require that the obtained blood culture become detectably positive (which takes 10 hours or more), followed by a sub-culturing step (at least 12 hours), and then an AST test (which requires a minimum of eight hours): this amounts to over forty-eight hours, depending on the pathogen, and often requires more than three days in practice. In most workflows, identification of organisms occurs after a sub-cultivation step and is increasingly performed by mass spectrometry. This delay in AST results directly extends the duration of broad-spectrum antimicrobial therapy as clinicians or pharmacists need to identify and AST results to prescribe an appropriate targeted antimicrobial. Furthermore, one shift operation, common in many clinical microbiology laboratories, often complicates waiting.

Alternatively, the invention can be used directly from positive blood cultures. After standard steps 1 and 2 of blood draw and incubation/culture, if the culture is positive, the culture flask will be moved directly to the microbial isolation (step 3) and then into an automated system (step 4). The present invention can be fully automated, requiring a technician to load the system with only a standard cassette with microbial dilution, and then initiate a 4 hour "quick-AST" process. The laboratory technician will then receive the same standard phenotypic results for AST at the minimum inhibitory concentration ("MIC"). However, the streamlined process can theoretically reduce the time to AST by more than twenty-four hours, and possibly by two days in practice, and simplify the laboratory workflow.

The AST systems currently in use perform a variation of the Clinical Laboratory Standards Institute (CLSI) liquid medium microdilution procedure. Bacteria are inoculated in parallel into a plurality of wells, each well containing a known concentration(s) of an antimicrobial agent and a nutrient broth. At 5X 10 ⁵ CFU/ml inoculation wells to ensure that the bacteria are in log phase growth are important for detecting accurate responses to the antimicrobial agent. The microbiological detection is then carried out visually.

The slow rate of the phenotypic AST test is due in part to its dependence on microbial growth to produce a detectable optical signal. Passing through less than-1 x10 ⁸ Densitometric measurement of the concentration of CFU/ml does not quantify the bacteria, making the CLSI starting concentration invisible for a minimum of eight times the time. Since the differentiation of microbial growth in slowest growing wells from microbial growth in non-growing wells is critical for Minimum Inhibitory Concentration (MIC) determination, significantly longer times are required. As known to those skilled in the art, some existing platforms overcome these growth problems by including metabolic probes in the liquid in which the microorganisms are suspended during incubation. However, the inclusion of these probes may miss the growth regime, such as filamentous growth, and may affect the accuracy of the results.

Once the AST-specific step has been initiated, the AST systems currently in use still typically require more than 8 hours to report results for simple, highly sensitive bacteria and more than 10 hours for pathogens with complex resistance profiles or slow growth kinetics, see fig. 4.

Furthermore, the automated AST systems currently in use suffer from two drawbacks that prevent accurate results from being reported in less than six hours: 1) Very major errors, inaccurate "sensitive" calls for truly resistant strains; and 2) major errors, inaccurate "resistance" calls for truly sensitive strains. Indeed, these problems have required BioMerieux and BD to modify their first 4 hour speed requirements of Vitek2 and Phoenix.

The presence of very major errors is explained in part by the metabolic energy consumed by the microorganisms in achieving antimicrobial resistance. Resistant microorganisms can alter energy expenditure in response to antimicrobial agents, confounding the results of metabolic probes around the MIC. These may also be caused by the presence of additives in the growth medium, such as redox indicators. The prevalence of major errors is mainly due to filamentous growth of certain bacteria. This mode of growth is a common antimicrobial response in gram-negative bacteria, particularly to cell wall acting antimicrobials such as beta-lactams. Filamentous bacteria continue to replicate their internal contents, but do not membrane. Thus, the metabolic probes again produced erroneous near MIC results. Removal of filamentous bacteria was shown to significantly reduce the major errors of the AST process.

The measurement of relative microbial surface area as used in the present invention overcomes the deficiencies of metabolic probes for AST. First, fast-AST enables fast and accurate resistance calling since changes in metabolic activity do not confound relative surface area. Second, surface area measurements prevent excessive resistance calling. The surface area measurement enables accurate discrimination between true resistance and filamentous growth compared to the volume measurement obtained with the metabolic probes of the AST systems currently in use. As illustrated in the schematic diagram of fig. 39, it is difficult to distinguish between the volumes of resistant and sensitive filamentous bacteria. The lack of separation makes the filamentous surface area significantly lower than that of the actual resistant bacteria. Thus, by amplifying the surface area of each bacterium, the present invention was able to accurately recall the 4 hour β -lactam (ampicillin) MIC of E.coli samples (see examples below). As illustrated in fig. 39, the difference in surface area between the elongated and "true" resistance is close to 2/3, which can be detected with an amplified signal.

Patient's health

As used herein, the term "patient" (also interchangeably referred to as "host" or "subject") refers to any host that can serve as a source of one or more biological samples or specimens discussed herein. In certain aspects, the donor will be a vertebrate, which is intended to mean any animal species (and preferably, mammalian species, such as humans). In certain embodiments, "patient" refers to any animal host, including, but not limited to, humans and non-human primates, avians, reptiles, amphibians, bovines, canines, caprines, cavities, crows, epines, equines, felines, caprines (hircins), lapines, hare (leporines), wolfes (lupines), ovines (ovines), porcines (porcins), racines, foxes (vulpines), and the like, including, but not limited to, domesticated livestock, herding or migratory animals or birds, exotic species or animal specimens, as well as companion animals, pets, and any animal under the care of a veterinarian.

Biological sample

A biological sample is any sample containing microorganisms (e.g., bacterial and fungal cells).

Exemplary biological samples include, but are not limited to, whole blood, plasma, serum, sputum, urine, stool, white blood cells, red blood cells, buffy coat, tears, mucus, saliva, semen, vaginal fluid, lymph fluid, amniotic fluid, spinal or cerebrospinal fluid, peritoneal exudate, pleural exudate, punctate material, epithelial smears, biopsy samples, bone marrow samples, fluid from cysts or abscesses, synovial fluid, vitreous or aqueous humor, eye wash or aspirate, bronchoalveolar lavage, bronchial lavage or pulmonary lavage, lung aspirate, and organs and tissues including, but not limited to, liver, spleen, kidney, lung, intestine, brain, heart, muscle, pancreas, and the like, swabs (including, but not limited to, wound swabs, oral swabs, throat swabs, vaginal, urethral swabs, cervical swabs, rectal swabs, lesion swabs, nasopharyngeal swabs, and the like), and any combination thereof. Also included are bacterial cultures or bacterial isolates, fungal cultures or fungal isolates. The skilled artisan will also appreciate that isolates, extracts, or materials obtained from any of the above exemplary biological samples are also within the scope of the present invention.

Microorganisms obtained from biological samples can be cultured or otherwise processed as is conventional in the art.

Exemplary microorganisms

As used herein, infection is intended to include any infectious agent of microbial origin, such as bacteria, fungal cells, archaea, and protozoa. In a preferred example, the infectious agent is a bacterium, such as a gram-positive bacterium, a gram-negative bacterium, and an atypical bacterium. The term "antimicrobial resistant microorganism" is a microorganism (e.g., bacteria, fungi, archaea, and protozoa) that is resistant to one or more different antimicrobial agents (i.e., antibacterial drugs, antifungal drugs, anti-archaea drugs, and anti-protozoan drugs).

The microorganism (e.g., a liquid suspension of microorganisms) may comprise a strain of microorganism. The microorganism may comprise a species of microorganism. The microorganism may comprise more than one strain of microorganism. The microorganism may comprise a microorganism of interest. The microorganism may comprise a class of microorganisms. The microorganism may comprise a microorganism of a family. The microorganism may comprise a microorganism of one kingdom.

The microorganism (e.g., a liquid suspension of microorganisms) may include more than one strain of microorganism. The microorganism may comprise more than one species of microorganism. The microorganism may comprise more than one species of microorganism. The microorganism may comprise more than one microorganism of interest. The microorganism may comprise more than one class of microorganism. The microorganisms may include microorganisms of more than one family. The microorganism may comprise more than one kingdom of microorganism.

The microorganism may be a bacterium. Examples of bacteria include, but are not limited to, acetobacter aureofaciens (A: (A))Acetobacter aurantius) Acinetobacter asphaltum: (A)Acinetobacter bitumen) Acinetobacter bacterium (A), (B)Acinetobacter spp.) Actinomycetes of Israel: (Actinomyces israelii) Actinomycetes genus (A)Actinomyces spp.) Genus Aerococcus (Aerococcus spp.) Agrobacterium radiobacter (A. Radiobacter) (B. Radiobacter)Agrobacterium radiobacter) Agrobacterium tumefaciens (A. Tumefaciens) (B)Agrobacterium tumefaciens) "amorphous", "amorphousAnaplasma) Phagocytophilic cell anaplasma (Anaplasma phagocytophilum) Rhizomatoid nitrogen-fixing rhizobia (A)Azorhizobium caulinodans) Azotobacter vinlandii (A)Azotobacter vinelandii) Bacillus bacteria (b), (b)Bacillus) Bacillus anthracis (B.), (Bacillus anthracis) Bacillus pumilus (B.pumilus) (B.pumilus)Bacillus brevis) Bacillus cereus (B.cereus)Bacillus cereus) Clostridium bacteria: (Bacillus fusiformis) Bacillus licheniformis: (A), (B)Bacillus licheniformis) Bacillus megaterium (B.), (Bacillus megaterium) Bacillus mycoides (B.) (Bacillus mycoides) Bacillus genus (A), (B)Bacillus spp.) Bacillus stearothermophilus: (A), (B)Bacillus stearothermophilus) Bacillus subtilis (B.subtilis) (B.subtilis)Bacillus subtilis) Bacillus thuringiensis (Bacillus thuringiensis) ((B))Bacillus Thuringiensis) Bacteroides (A) and (B)Bacteroides) Bacteroides fragilis: (A), (B)Bacteroides fragilis) Bacteroides gingivalis: (A. Gingivalis)Bacteroides gingivalis) Bacteroides melanogenes: (A. Melanogenes:)Bacteroides melaninogenicus) (also known as melanogenesis Prevotella: (Prevotella melaninogenica) Baltone's body (B) ((B))Bartonella) Bartonella handii (B)Bartonella henselae) Bartonella body of five sunscals: (Bartonella quintana) Bartonella (a. Bor.) (Bartonella spp.) Bordet, bordet bacteria (A), (B)Bordetella) Bordetella bronchiseptica (Bordetella bronchiseptica)Bordetella bronchiseptica) Bordetella pertussis (B.pertussis)Bordetella pertussis) Bordetella (B, C)Bordetella spp.) Borrelia burgdorferi (B)Borrelia burgdorferi) Brucella (Brucella) (Brucella)Brucella) Brucella abortus: (Brucella abortus) Brucella melitensis (C.)Brucella melitensis) Brucella (Brucella) (Brucella)Brucella spp.) Brucella suis (Brucella suis) ((R))Brucella suis) Berkohall, a.k.a. Dermata bacteria (A), (B)Burkholderia) Burkholderia cepacia (B.)Burkholderia cepacia) Boke of deep rooted carbuncle of nose Hold. Bacterium (B) ((C))Burkholderia mallei) Burkholderia pseudorhinis: (B.pseudorhinis) (C.pseudorhinis)Burkholderia pseudomallei) Sheath rod bacterium of granulomatous bacteria (A), (B)Calymmatobacterium granulomatis) Campylobacter bacteria (A), (B)Campylobacter) Campylobacter coli: (Campylobacter coli) Campylobacter fetus: (Campylobacter fetus) Campylobacter jejuni: (Campylobacter jejuni) Campylobacter pylori: (Campylobacter pylori) Campylobacter genus (A)Campylobacter spp.) Chlamydia (C.), (B.), (C.), (C.Chlamydia) Chlamydia genus (A)Chlamydia spp.) Chlamydia trachomatis (C)Chlamydia trachomatis) Chlamydophila (Chlamydophila) Chlamydophila pneumoniae: (Chlamydophila pneumoniae) (previously known as Chlamydia pneumoniae: (Chlamydia pneumoniae) Chlamydophila psittaci ((iii)), (ii)Chlamydophila psittaci) (previously known as Chlamydophila psittaci: (Chlamydia psittaci) Genus Chlamydophila: (C)Chlamydophila spp.) Clostridium difficile (ii) (Clostridium) Clostridium botulinum (C.) (Clostridium botulinum) Difficile of Clostridium difficile: (C. Difficile)Clostridium difficile) Clostridium perfringens (A)Clostridium perfringens) (previously known as Clostridium welchii: (Clostridium welchii) Clostridium (A), (B) and (B)Clostridium spp.) Clostridium tetani (C. Tetani)Clostridium tetani) Corynebacterium bacterium (C.sp.), (Corynebacterium) Corynebacterium diphtheriae (C.), (Corynebacterium diphtheriae) Corynebacterium fusiform (C.sp.), (Corynebacterium fusiforme) Corynebacterium genus (A)Corynebacterium spp.) Burkholderia (B.C.)Coxiella burnetii) Chaffeensis (E.chaffeensis)Ehrlichia chaffeensis) Genus Escherichia (A), (B), (C)Ehrlichia spp.) Enterobacter cloacae: (Enterobacter cloacae) Enterobacter genus (A)Enterobacter spp.) Enterococcus bacteria (A)Enterococcus) Enterococcus avium (C.) (Enterococcus avium) Durable intestinal ballBacteria (A), (B)Enterococcus durans) Enterococcus faecalis: (Enterococcus faecalis) Enterococcus faecium (C. Faecium)Enterococcus faecium) Enterococcus gallinarum (F) and (F)Enterococcus galllinarum) Enterococcus malodoratus (C.), (Enterococcus maloratus) Enterococcus genus (C.) (Enterococcus spp.) Escherichia coli (E.coli)Escherichia coli) Francisella (Francisella)Francisella spp.) Francisella tularensis (A.terrestris)Francisella tularensis) Fusobacterium nucleatum (A)Fusobacterium nucleatum) Gardnerella (Gardner) genus (Gardenerella spp.) Gardnerella vaginalis (A. Vaginalis) ((B. Vaginalis))Gardnerella vaginalis) Haemophilus genus (A), (B), (C)Haemophilius spp.) Haemophilus bacteria (C.), (Haemophilus) Haemophilus ducreyi: (Haemophilus ducreyi) Haemophilus influenzae: (Haemophilus influenzae) Haemophilus parainfluenza: (Haemophilus parainfluenzae) Bordetella pertussis: (B.pertussis: (B.pertussis)Haemophilus pertussis) Haemophilus vaginalis (A), (B), (C)Haemophilus vaginalis) Helicobacter pylori (Helicobacter pylori) Helicobacter species (A) and (B)Helicobacter spp.) Klebsiella pneumoniae (K.pneumoniae)Klebsiella pneumoniae) Klebsiella genus (A), (B), (C)Klebsiella spp.) Lactobacillus (b), (b)Lactobacillus) Lactobacillus acidophilus (Lactobacillus acidophilus) Lactobacillus bulgaricus (A. Borealis: (A.))Lactobacillus bulgaricus) Lactobacillus casei (L.casei) (L.) C.Lactobacillus casei) Lactobacillus (A), (B) and (C)Lactobacillus spp.) Lactococcus lactis: (Lactococcus lactis) Legionella pneumophila (Legionella pneumophila) Legionella (Legionella) (Legionella)Legionella spp.) The fine helix belongs to (Leptospira spp.) Listeria monocytogenes: (A), (B)Listeria monocytogenes) Listeria genus (a)Listeria spp.)，extroquenMethanobacterium bacterium (A), (B)Methanobacterium extroquens) Microbacterium polymorpha (A), (B), (C)Microbacterium multiforme) Micrococcus luteus (A) and (B)Micrococcus luteus) Moraxella catarrhalis: (Moraxella catarrhalis) Mycobacterium: (A), (B)Mycobacterium) Mycobacterium avium: (Mycobacterium avium) Mycobacterium bovis: (Mycobacterium bovis) Mycobacterium diphtheriae (I), (II)Mycobacterium diphtheriae) M. intracellulare (M.intracellulare) ((Mycobacterium intracellulare) Mycobacterium leprae (Ma.), (Mycobacterium leprae) Mycobacterium leprae (M.murinus) ((M.murinus))Mycobacterium lepraemurium) Mycobacterium phlei: (Mycobacterium phlei) Mycobacterium smegmatis: (Mycobacterium smegmatis) Mycobacterium genus (A), (B), (C)Mycobacterium spp.) Mycobacterium tuberculosis: (Mycobacterium tuberculosis) Mycoplasma (A) and (B)Mycoplasma) Fermenting Mycoplasma (M.) (Mycoplasma fermentans) Mycoplasma genitalium (Mycoplasma genitalium) Human mycoplasma (A. Hominis) ((B))Mycoplasma hominis) Penetrating mycoplasma (Mycoplasma penetrans) Mycoplasma pneumoniae (A)Mycoplasma pneumoniae) The genus of dendron (Mycoplasma spp.) Neisseria bacterium (N.neisseria bacterium) ((Neisseria) N. gonorrhoeae (N.gonorrhoeae) ((S))Neisseria gonorrhoeae) N. meningitidis (N.meningitidis: (Neisseria meningitidis) Neisseria genus (N)Neisseria spp.) Nocardia genus (A)Nocardia spp.) Pasteurella (a) and (b)Pasteurella) Pasteurella multocida (A), (B), (C)Pasteurella multocida) Pasteurella (a) and (b)Pasteurella spp.) Pasteurella terrestris (A)Pasteurella tularensis) Digestive streptococcus (C.) (Peptostreptococcus) Porphyromonas gingivalis: (Porphyromonas gingivalis) Privo bacterium (a) producing melaninPrevotella melaninogenica) (previously known as melanogenesis Bacteroides: (Bacteroides melaninogenicus) Proteobacteria (A), (B) and (C)Proteus spp.) Pseudomonas aeruginosa (Pseudomonas aeruginosa) ((Pseudomonas aeruginosa) Pseudomonas (A) or (B)Pseudomonas spp.) Rhizobium radiobacter (A)Rhizobium radiobacter) Rickettsia (a)Rickettsia) Rickettsia prowazekii (Rickettsia prowazekii) Psittacosis rick hypo-structure of the body (A), (B), (C)Rickettsia psittaci) Five-day fever rickettsia (Rickettsia quintana) Rickettsia, rickettsia (a)Rickettsia rickettsii) Genus Rickettsia (a)Rickettsia spp.) "Shaoyelike" body (a. Thaliana)Rickettsia trachomae) Rocarby martensite (c) ((c))Rochalimaea) Rockwell martensite (C)Rochalimaea henselae) Five daysHot rocarbli martensite (Rochalimaea quintana)，Rothia dentocariosaSalmonella bacteria (A), (B)Salmonella) Salmonella enteritidis (Salmonella enteritidis) Salmonella genus (A), (B)Salmonella spp.) Salmonella typhi (A), (B)Salmonella typhi) Salmonella typhimurium (Salmonella typhimurium) Serratia marcescens (A), (B), (C)Serratia marcescens) Shigella dysenteriae: (A), (B), (C)Shigella dysenteriae) Shigella (A), (B) and (C)Shigella spp.) Spiro bacteria (S.), (Spirillum volutans) Staphylococcus (1)Staphylococcus) Staphylococcus aureus (S.) (Staphylococcus aureus) Staphylococcus epidermidis: (Staphylococcus epidermidis: (Staphylococcus epidermidis) Staphylococcus genus (S.aureus)Staphylococcus spp.) Stenotrophomonas maltophilia (Stenotrophomonas maltophilia) Stenotrophomonas genus (A), (B)Stenotrophomonas spp.) Streptococcus (S.) (Streptococcus) Streptococcus agalactiae (Streptococcus agalactiae) Bird streptococcus (Streptococcus avium) Streptococcus bovis (1)Streptococcus bovis) Streptococcus rat: (Streptococcus cricetus) Streptococcus faecium (I)Streptococcus faceium) Streptococcus faecalis: (A)Streptococcus faecalis) Wild streptococcus (C.), (Streptococcus ferus) Streptococcus gallinae: (Streptococcus gallinarum) Streptococcus lactis (C.lactis: (A.), (B.lactis)Streptococcus lactis) Streptococcus mitis: (S. Miltiorrhiae) (S. Mil)Streptococcus mitior) Streptococcus mitis (S. Mitis)Streptococcus mitis) Streptococcus mutans (A)Streptococcus mutans) Oral streptococci (Streptococcus oralis) Streptococcus pneumoniae (S.pneumoniae:Streptococcus pneumoniae) Streptococcus pyogenes: (Streptococcus pyogenes) Streptococcus murinus (C. Murinus)Streptococcus rattus) Streptococcus salivarius (S. Salivarius:Streptococcus salivarius) Streptococcus sanguis (S.) (Streptococcus sanguis) Streptococcus sorbinus (S.brothers) (S.brothers)Streptococcus sobrinus) Streptococcus genus (C)Streptococcus spp.) Treponema (Treponema) Treponema denticola (A)Treponema denticola) Treponema pallidum (A)Treponema pallidum) Treponema (R) genusTreponema spp.) Urocladia genus (Ureaplasma spp.) Vibrio (Vibrio)Vibrio) Vibrio cholerae (V. Cholerae) ((V. Cholerae))Vibrio cholerae) Vibrio comma (V.comma) ()Vibrio comma) Vibrio parahaemolyticus: (Vibrio parahaemolyticus) Genus Vibrio: (Vibrio spp.) Vibrio vulnificus (Vibrio vulnificus) Streptococcus viridis: (A. Viridis:)viridans streptococci) Wolbachia (W.W.)Wolbachia) Yersinia bacterium (Yersinia) ((R))Yersinia) Yersinia enterocolitica (yersinia enterocolitica) (ii)Yersinia enterocolitica) Yersinia pestis: (Yersinia pestis) Yersinia pseudotuberculosis: (Yersinia pseudotuberculosis) And Yersinia genus (Yersinia spp.)。

The microorganism may be a fungus. Examples of fungi include and is not limited to Aspergillus (A)Aspergillus spp.) Genus Germinatum: (Blastomyces spp.) Candida genus (A)Candida spp.) Cladosporium (A) and (B)Cladosporium) Genus coccidiodes (A)Coccidioides spp) Cryptococcus genus (Cryptococcus spp.) Genus Helminthosporium (II)Exserohilum) Fusarium genus (A)fusarium) Tissue of the same Histoplasma genus (A)Histoplasma spp.) Issatchenkia (a)Issatchenkia spp.) Mucor genus (A), (B), (C)mucormycetes) Pneumocystis genus (Pneumocystis spp.) Ringworm (ringworm), hyphomycete (Siberian)scedosporium) Sporomyces (A) and (B)Sporothrix) And Stachybotrys (Stachybotrys spp)。

The microorganism may be a protozoan. Examples of protozoa include, and are not limited to, entamoeba histolytica (Entamoeba histolytica) Plasmodium genus (A), (B), (C)Plasmodium spp.) Giardia lamblia (Giardia lamblia) (II)Giardia lamblia) And Trypanosoma brucei: (Trypanosoma brucei)。

Exemplary antimicrobial Agents

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Antimicrobial agents whose interaction with microorganisms affects the negative charge on the surface of the microorganisms and which are affected by the negative charge on the surface of the microorganisms include: polycationic aminoglycosides that displace Mg upon binding to the cell surface ²⁺ Ions that bridge the lipid membrane components, thereby disrupting the outer membrane and enhancing drug uptake; cationic polymyxins (colistin and polymyxin B), the binding of which to microbial cells is also dependent on the negative charge of the membrane and undergo mutation and plasmid-mediated resistance thereto by reducing the negative charge of the membrane; and daptomycin, a lipopeptide that is similar to a host innate immune response cationic antimicrobial peptide and requires Ca ²⁺ And phosphatidylglycerol as their mechanism of action to disrupt membranes, and resistance to them can also involve changes in cell surface charge.

When the microorganism is a fungus, exemplary antimicrobial agents include 5-flucytosine, abafungin, abaconazole, allylamines, amphotericin B, ancobon, anidulafungin, oxazole, peruvian balsam, benzoic acid, bifonazole, butoconazole, candida, caspofungin, ciclopirox, clotrimazole, cresemba, crystal violet, sulbactam, echinocandins, econazole, effluconazole, epoxiconazole, fenticonazole, filipin, fluconazole, flucytosine, griseofulvin, gris-Peg, haloprogin, hamycin, imidazoles, isavuconazole, isazolium, isoconazole, itraconazole, ketoconazole, sulconazole, micafungin, miconazole, natamycin, noccoffeifen, nystatin, omoconazole, ormavig, orvavir, oxconazole, posaconazole, propiconazole, raconazole, lansoproconazole, thioconazole, fenticonazole, thiofenticonazole, fenamidothioconazole, fenamidothiofenamate, and derivatives thereof.

When the microorganism is a protozoan, exemplary antimicrobial agents include 8-aminoquinoline, arsanilide, agents directed against phylum proteorum, ailanthone, amodiaquine, amphotericin B, aprrolium, an anti-trichomonal agent, malamycin, arsothinol, artelinic acid, artemether/lumefantrine, artemisinin, arteether, artexane, artesunate/amodiaquine, atovaquone/proguanil, azanidazole, azithromycin, benznidazole, bromohydroxyquinoline, buparvaquone, carbaarsine, ornidazole, quiniodine, chloroquine, chlorpromoguanide/dapsone, proguanil/dapsone/artesunate, cloquindol, cloquintocet, clomazone antiparasitics, cinchona, cipargamin, clazuril, clifam, keqingnor, coccidium inhibitors, codinaeopsin, cotrifazid, sinomenine, cycloguanidine, dehydroemidine, diphenylarsine, dihydroartemisinin, dichloronitle, diazoamazamide, disulfiram, doxycycline, elQ-300, emidine, etodoxa, fumarin, furazoxane, nagearsine, GNF6702, halofantrine, hydroxychloroquine medocarb, ipronidazole, jesuit's bark, KAF156, lumefantrine, maduramicin, mefloquine, megazol, meglumine antimonate, melarsanol, mepacrine, metronidazole, miltefosine, neurolenin B, nicarbazin, nifurolimus, nimorazole, nitraarsonic acid, nitidine, nifuratel, olivacine, ornidazole, oroidin, pamaquine, paromomycin, pentamidine, pentavalent antimony, ubiquinone, oxydiphenylamidine, piperaquine, primaquine, proguanil, item 523, pronidazole, jenuride, etc, ethirime, pyronaridine, quinfamide, quinine, ronidazole, schediula Romana, SCYX-7158, secnidazole, samimod, sodium antimony gluconate, spirocyclic indolone, sulfadoxine-ethirime, sulfalene, suramin, tafluoroquine, tenolol, tenonazole, mebroquinol (Tilbroquinol), tinidazole, trimetrexate, trypanoside, warburg tincture, and a generalized form or variant thereof.

The antimicrobial agent may be a drug that acts by a mechanism similar to the drugs described herein.

Other antimicrobial agents known in the art may be used in the present invention.

Liquid suspension

The liquid suspension of microorganisms may be agitated by the use of mechanical, acoustic and/or magnetic agitation. Examples of mechanical agitation include shaking or shaking and/or the use of a stir bar, paddle, stirring blade and/or propeller or impeller.

The microbial isolation is carried out by: centrifuging (e.g. in order tog-force>500 × g) Magnetic separation, filtration (e.g., via a filter having pores less than or equal to 0.45 microns, and preferably less than or equal to 0.2 microns), electrophoresis, dielectrophoresis, precipitation, agglutination, or any combination thereof.

The liquid may include a growth medium, such as cation-regulated Mueller Hinton liquid medium. The medium may contain one or more additives known to those skilled in the art to promote the growth and stability of microorganisms. In addition to different antimicrobial agents, different test wells may contain one or more additives known to improve AST accuracy of a particular antimicrobial agent. For example, additional sodium chloride may be added to a test comprising oxacillin, and additional calcium may be added to a test comprising daptomycin.

Box

The type of cassette is not limited. A cartridge is a container capable of holding and allowing the growth of a liquid suspension of microorganisms. Non-limiting examples of cassettes include culture bottles, petri dishes, bioassay dishes, culture tubes, test tubes, microcentrifuge tubes, bottles, microplates, multiwell plates, microtiter plates, and microwell plates. The cartridge may contain a chamber. The cartridge may comprise a plurality of chambers, each chamber being a space capable of holding the liquid suspension physically separate from another space; one example of a chamber is a well in a multiwell plate. The cartridge may contain 1,2,3, 4,5, 6,7, 8,9, 10, 11, 12, 24, 48, 96, 192, 384, 1536 or more chambers, and any number of chambers therebetween.

Optical device

Any optical device (e.g., microscope, microplate reader) capable of detecting a signal having a number of different characteristics may be used in the present invention. For example: broad spectrum lamps (e.g., xenon), narrow spectrum lamps, lasers, LEDs, multiphoton, confocal, or total internal reflection illumination may be used for excitation. Cameras (single or multiple), single photodiodes or arrays of photodiodes (ID or 2D), avalanche photodiodes, CMOS or CCD sensors, solid state photomultipliers (e.g. silicon photomultipliers) and/or photomultiplier (single or multiple) with filter-based or grating-based spectral resolution (one or more spectrally resolved emission wavelengths) are possible on the detection side.

Reagent kit

The terms "kit" and "system" as used herein in the present invention mean a combination of such things as a plurality of signaling agents with one or more other types of elements or components (e.g., other types of biochemical reagents, signal detection reagents, controls (i.e., positive and negative controls, e.g., chemosensitive/resistant microorganisms), means of separation (e.g., filters and magnetic beads), containers, packaging such as packaging for commercial sale, substrates/cartridges in which suspensions of microorganisms can be cultured, processed, or contained, electronic hardware components, and software recorded on a non-transitory processor readable medium).

Another aspect of the invention is a kit for determining antimicrobial sensitivity of a microorganism. The kit comprises a signaling agent capable of binding to the surface of the target intact microorganism; a solution for incubating a sample containing a microorganism; and one or more agents for generating a signal from the signaling agent.

In embodiments, the signaling agent is associated with one or more binding moieties capable of binding, directly or indirectly, to the target intact microorganism.

In embodiments, the one or more binding moieties are selected from the group consisting of antibodies, lectins, natural and/or synthetic peptides, synthetic and/or natural ligands, synthetic and/or natural polymers, synthetic and/or natural glycopolymers, carbohydrate-binding proteins and/or polymers, glycoprotein-binding proteins and/or polymers, charged small molecules, other proteins, bacteriophages and/or aptamers.

In embodiments, the one or more binding moieties may be polyclonal and/or monoclonal antibodies.

In embodiments, the one or more binding moieties may be synthetic and/or natural ligands and/or peptides. The ligand and/or peptide may be selected from bis (zinc-lutidine), TAT peptide, serine protease, cathelicidins, cationic dextrin, cationic cyclodextrin, salicylic acid, lysine and combinations thereof.

In embodiments, the one or more binding moieties may be synthetic and/or natural polymers and/or sugar polymers. The natural and/or synthetic polymer may be pullulan, poly (N- [3- (dimethylamino) propyl ] methacrylamide), poly (ethyleneimine), poly-L-lysine, poly [ 2- (N, N-dimethylamino) ethyl methacrylate ], and combinations thereof. The natural and/or synthetic polymers and/or sugar polymers may include moieties including, but not limited to, chitosan, gelatin, dextran, trehalose, cellulose, mannose, cationic dextrans and cyclodextrins (cyclodextrans) or combinations thereof including, but not limited to, co-block, graft and alternating polymers.

In embodiments, the one or more binding moieties may comprise a glycoprotein selected from the group consisting of mannose binding lectins, other lectins, annexins, and combinations thereof.

In embodiments, the one or more binding moieties may comprise two or more binding moieties.

In embodiments, the one or more binding moieties may directly or indirectly bind to one or more biomolecules present on the surface of the microorganism. Exemplary biomolecules include peptidoglycans, mureins, mannoproteins, porins, beta-glucans, chitins, glycoproteins, polysaccharides, lipopolysaccharides, lipooligosaccharides, lipoproteins, endotoxins, lipoteichoic acids, teichoic acids, lipid a, carbohydrate binding domains, efflux pumps, other cell wall and/or cell membrane associated proteins, other anionic phospholipids, and combinations thereof.

In embodiments, the binding moiety is a nanoparticle.

In embodiments, the binding moiety is a bacteriophage.

In embodiments, the one or more binding moieties may specifically bind to one or more biomolecules.

In embodiments, the one or more binding moieties may bind to one or more species-specific biomolecules.

In embodiments, the one or more binding moieties may non-specifically bind to the microorganism, e.g., via non-covalent interactions and via formation of covalent bonds.

In embodiments, the kit further comprises magnetic beads to magnetically separate the microorganisms from the supernatant.

In embodiments, the magnetic beads are associated with one or more binding moieties that bind to a microorganism. The one or more binding moieties associated with the magnetic beads can be the same as those associated with the signaling agent. The one or more binding moieties associated with the magnetic beads can be different from those associated with the signaling agent.

In embodiments, the magnetic beads have a diameter in the range of 20nm to 20 microns.

In embodiments, the kit further comprises one or more ions or small molecules to enhance binding between the binding moiety and the microorganism.

In embodiments, the solution comprises < 0.15M salt.

In embodiments, the kit further comprises a microbial binding agent, and wherein the binding moiety indirectly binds the microorganism via the microbial binding agent. The binding moiety may be conjugated to streptavidin, neutravidin or avidin, and the microbial binding agent may be biotinylated. The binding moiety may be an antibody that binds to a species-specific Fc domain, and the microbial binding agent may be an antibody capable of binding to a microorganism with a species-specific Fc domain.

In embodiments, the signaling agent may include one or more of a chemiluminescent group, a catalyst, or an enzyme. The enzyme may be at least one of horseradish peroxidase, alkaline phosphatase, acetylcholinesterase, glucose oxidase, beta-D-galactosidase, beta-lactamase, and a combination thereof. The catalyst may be an organometallic compound.

In embodiments, the signaling agent is provided in the form of a nanoparticle, e.g., the signaling agent is encapsulated within the nanoparticle. The nanoparticles may be dissociable, which may include a metal oxide; the metal oxide may be or include iron oxide, cesium oxide and/or cerium oxide.

In embodiments, zero, one or two washes are performed prior to determining the signal level.

In embodiments, zero, one, or two washes are performed prior to addition of the signaling agent.

In embodiments, the kit further comprises a color reagent to generate a measurable signal.

In embodiments, the one or more reagents include a reagent for catalyzing a reaction and a reagent for stopping a catalyzed reaction.

In embodiments, the kit further comprises a means for measuring a signal (e.g., an optical and/or electrical signal). The optical measurements may be fluorescent, time-resolved fluorescent, absorptive and/or luminescent.

In embodiments, the kit further comprises a multiwell plate, such as a 24-well, 96-well, 192-well, or 384-well plate.

In embodiments, the kit further contains instructions for using the kit to perform the methods disclosed herein. The kit may additionally contain instructions for performing the steps performed before or after one or more of the methods described herein.

In one embodiment, kits are provided that contain reagents necessary to perform one or more methods as described herein or reagents necessary to perform steps before or after one or more methods as described herein.

Method of treatment

As used herein, the terms "treat", "treating", "treatment", and the like refer to reducing or ameliorating a disorder and/or symptoms associated therewith. It is understood that, although not excluded, treating a disorder or condition does not require complete elimination of the disorder, condition, or symptoms associated therewith. Treatment may include a healthcare professional or diagnostic scientist recommending a desired course of action or treatment, e.g., a prescription, to the subject.

As used herein, the terms "prevent", "preventing", "prophylactic treatment", and the like refer to reducing the likelihood of developing a disorder or condition in a subject who is not at risk of developing the disorder or condition or who is susceptible to developing the disorder or condition.

The term "method of treatment" includes methods of management and, when used in conjunction with an infectious microbial organism or infection, includes ameliorating, eliminating, reducing, preventing, or otherwise alleviating or managing the deleterious effects of an infectious microbe or of an infectious microbe.

As used herein, the terms "drug", "therapeutic", "active agent", "therapeutic compound", "composition", or "compound" are used interchangeably and refer to any chemical entity, drug (pharmaceutical), drug (drug), biological, botanical, etc., that can be used to treat or prevent a disease, condition, or disorder of a bodily function. The medicament may comprise a known and potentially therapeutic compound. The drug may be determined to be therapeutic by screening using screens known to those of ordinary skill in the art. "known therapeutic compound", "drug" or "drug" refers to a therapeutic compound that has been shown (e.g., by animal testing or prior experience of administration to humans) to be effective in such treatment. A "treatment regimen" relates to a treatment comprising a "drug", "therapeutic agent", "active agent", "therapeutic compound", "composition" or "compound" as disclosed herein and/or a treatment comprising a behavioral modification of a subject and/or a treatment comprising a surgical means.

Antimicrobial agents, such as antibiotics, are agents that are capable of killing or inhibiting the growth of microorganisms.

Signal transduction Agents and chemical ligation

In embodiments, the invention features a signaling agent capable of binding to a surface of a microorganism. In embodiments, the binding is non-specific. In embodiments, the binding is specific.

In embodiments, the signaling agent is present during the incubation step of the methods described herein. In embodiments, the signaling agent is present after the incubation step of the methods described herein.

In embodiments, binding comprises forming a covalent bond. In embodiments, the signaling agent is capable of binding to the surface of the microorganism, wherein said binding comprises forming a covalent bond. In embodiments, a method as described herein results in the formation of a covalent bond between a group (e.g., via a reactive group, such as an electrophilic or nucleophilic group as described herein) on a microbial surface and a signaling agent as described herein. In embodiments, the signaling agent has formed a covalent bond with the surface of the microorganism.

In embodiments, binding comprises forming a non-covalent interaction. In embodiments, the signaling agent is capable of binding to a surface of a microorganism, wherein said binding comprises forming a non-covalent interaction. In embodiments, a method as described herein results in the formation of a non-covalent interaction between a group on a microbial surface (e.g., via a reactive group, such as an electrophilic or nucleophilic group as described herein) and a signaling agent as described herein. In embodiments, the signaling agent has formed a non-covalent interaction with the surface of the microorganism.

In embodiments, the non-covalent interaction comprises: ionic interactions, ion-ion interactions, dipole-dipole interactions, ion-dipole interactions, electrostatic interactions, london dispersion, van der waals interactions, hydrogen bonding, pi-pi interactions, hydrophobic interactions, or any combination thereof. In embodiments, the non-covalent interaction is: ionic interactions, ion-ion interactions, dipole-dipole interactions, ion-dipole interactions, electrostatic interactions, london dispersion, van der waals interactions, hydrogen bonding, pi-pi interactions, hydrophobic interactions, or any combination thereof.

In embodiments, the non-covalent interactions comprise ionic interactions, van der waals interactions, hydrophobic interactions, pi-pi interactions, or hydrogen bonds, or any combination thereof. In embodiments, the non-covalent interactions comprise ionic interactions, van der waals interactions, hydrogen bonds, or pi-pi interactions, or any combination thereof.

In embodiments, the signaling agent capable of binding to the surface of a microorganism comprises a group (e.g., a chemical or biochemical group) capable of binding to a microorganism's membrane, wall, protein, organelle, sugar, lipid, cell envelope, or nucleic acid, or any combination thereof. In embodiments, the signaling agent capable of binding to the surface of a microorganism comprises a chemical group (e.g., a nucleophilic group or an electrophilic group) capable of binding to a microbial membrane, wall, protein, organelle, sugar, lipid, cell envelope, or nucleic acid, or any combination thereof. In embodiments, the signaling agent capable of binding to the surface of a microorganism comprises a biochemical group capable of binding to a microorganism membrane, wall, protein, organelle, sugar, lipid, cell envelope, or nucleic acid, or any combination thereof.

In embodiments, the surface may comprise a biomolecule to which the signaling agent is bound or associated. Exemplary biomolecules include peptidoglycans, mureins, mannoproteins, porins, beta-glucans, chitins, glycoproteins, polysaccharides, lipopolysaccharides, lipooligosaccharides, lipoproteins, endotoxins, lipoteichoic acids, teichoic acids, lipid a, carbohydrate binding domains, efflux pumps, other cell wall and/or cell membrane associated proteins, other anionic phospholipids, and combinations thereof.

In embodiments, the signaling agent capable of binding to the surface of a microorganism comprises a biochemical group capable of binding to a microorganism membrane, wall, protein, organelle, sugar, lipid, cell envelope, or nucleic acid, or any combination thereof.

In embodiments, the signaling agent capable of binding to the surface of a microorganism comprises a chemical group (e.g., a nucleophilic functional group or an electrophilic functional group) capable of binding to a microbial membrane, wall, protein, organelle, sugar, lipid, cell envelope, or nucleic acid, or any combination thereof. In embodiments, the chemical group is a nucleophilic functional group. In embodiments, the chemical group is an electrophilic functional group.

In embodiments, the signaling agent is a biochemical signaling agent. In embodiments, the biochemical signaling agent comprises a biomolecule, such as an antibody, ligand, protein, aptamer, ss-DNA, ss-RNA, or ss-PNA).

In embodiments, the signaling agent is a chemical signaling agent. In embodiments, the chemical signaling agent is a chemical compound (e.g., a synthetic chemical compound). In embodiments, the chemical signaling agent does not comprise a biomolecule, such as an antibody, ligand, protein, aptamer, ss-DNA, ss-RNA, or ss-PNA).

In embodiments, the signaling agent capable of binding to the surface of a microorganism comprises

A linker group L; and

an amplifier group (e.g., amplifier group 104 as a chemical or biochemical amplifier).

In an embodiment, the amplifier group is an amplifier group 104, which is a chemical or biochemical amplifier. In an embodiment, the amplifier group 104 is a chemical amplifier. In an embodiment, the amplifier group 104 is a biochemical amplifier.

In embodiments, the signaling agent is a chemical compound. In embodiments, the chemical compound comprises a chemical amplifier group, such as those described herein.

In embodiments, the linker group L comprises a conserved (Fc) region of an antibody.

In embodiments, the linker group L is capable of forming a covalent bond with an amplifier group (e.g., amplifier group 104 as a chemical or biochemical amplifier).

In embodiments, the linker group L forms a covalent bond with a signal amplifier group (e.g., amplifier group 104 as a chemical or biochemical amplifier).

In embodiments, the linker group L is capable of forming one or more non-covalent interactions with an amplifier group (e.g., amplifier group 104 as a chemical or biochemical amplifier).

In embodiments, the linker group L forms one or more non-covalent interactions with an amplifier group (e.g., amplifier group 104 as a chemical or biochemical amplifier).

In embodiments, the linker group L comprises a group (e.g., a chemical or biochemical group) capable of binding to a microbial surface. In embodiments, the linker group L comprises a group (e.g., a chemical or biochemical group) that binds to the surface of the microorganism.

In embodiments, the linker group L comprises a group (e.g., a chemical or biochemical group) capable of forming a covalent bond with the surface of the microorganism. In embodiments, the linker group L comprises a group (e.g., a chemical or biochemical group) that forms a covalent bond with the surface of the microorganism.

In embodiments, the linker group L comprises a group (e.g., a chemical or biochemical group) capable of forming one or more non-covalent interactions with the surface of a microorganism. In embodiments, the linker group L comprises a group (e.g., a chemical or biochemical group) that forms one or more non-covalent interactions with the surface of the microorganism.

In embodiments, the linker group L comprises a chemical moiety 101, wherein the chemical moiety is capable of forming a non-covalent interaction with a surface of a microorganism. In embodiments, the linker group L comprises a chemical moiety 101, wherein said chemical moiety is capable of forming a covalent bond with the surface of a microorganism. In embodiments, the linker group L comprises a chemical moiety 101, wherein the chemical moiety forms a non-covalent interaction with the surface of the microorganism. In embodiments, the linker group L comprises a chemical moiety 101, wherein the chemical moiety forms a covalent bond with the surface of the microorganism.

In embodiments, linker group L comprises spacer moiety 102. In embodiments, spacer moiety 102 is covalently attached to chemical moiety 101 and/or to chemical moiety 103. In embodiments, spacer moiety 102 is covalently attached to chemical moiety 101. In embodiments, spacer moiety 102 is covalently attached to chemical moiety 103. In embodiments, spacer moiety 102 is covalently attached to chemical moiety 101 and to chemical moiety 103. In embodiments, spacer moiety 102 forms a non-covalent interaction with chemical moiety 101 and/or with chemical moiety 103. In embodiments, spacer moiety 102 forms a non-covalent interaction with chemical moiety 101. In embodiments, spacer moiety 102 forms a non-covalent interaction with chemical moiety 103. In embodiments, spacer moiety 102 forms a non-covalent interaction with chemical moiety 101 and with chemical moiety 103.

In embodiments, the linker group L comprises a chemical moiety 103, wherein the chemical moiety is capable of forming a covalent bond with an amplifier group (e.g., amplifier group 104 as a chemical or biochemical amplifier). In embodiments, the linker group L comprises a chemical moiety 103, wherein the chemical moiety has formed a covalent bond with an amplifier group (e.g., amplifier group 104 as a chemical or biochemical amplifier). In embodiments, the linker group L comprises a chemical moiety 103, wherein the chemical moiety is capable of forming a non-covalent interaction with an amplifier group (e.g., amplifier group 104 as a chemical or biochemical amplifier). In embodiments, the linker group L comprises a chemical moiety 103, wherein the chemical moiety has formed a non-covalent interaction with an amplifier group (e.g., amplifier group 104 as a chemical or biochemical amplifier).

In embodiments, the signaling agent is a chemical compound comprising a linker group L comprising:

a chemical moiety 101, wherein the chemical moiety is capable of forming a covalent bond or a non-covalent interaction with a surface of a microorganism;

a spacer moiety 102, wherein the spacer moiety is covalently linked to chemical moiety 101 and to chemical moiety 103; and

a chemical moiety 103, wherein said chemical moiety has formed or can form a covalent bond with an amplifier group 104 that is a chemical or biochemical amplifier.

In embodiments, the signaling agent is a chemical compound comprising a linker group L comprising:

a chemical moiety 101, wherein the chemical moiety is capable of forming a covalent bond or a non-covalent interaction with a surface of a microorganism;

a spacer moiety 102, wherein the spacer moiety is covalently linked to chemical moiety 101 and to chemical moiety 103; and

a chemical moiety 103, wherein said chemical moiety has or can form a non-covalent interaction with an amplifier group 104 that is a chemical or biochemical amplifier.

In embodiments, the linker group comprises one chemical moiety 101. In embodiments, the linker group comprises more than one chemical moiety 101 (e.g., the linker group comprises 1,2,3, 4,5, or 6 chemical moieties 101).

In embodiments, the linker group comprises one spacer moiety 102. In embodiments, a linker group comprises more than one spacer moiety 102 (e.g., a linker group comprises 1,2,3, 4,5, or 6 spacer moieties 102).

In embodiments, the linker group comprises one chemical moiety 103. In embodiments, a linker group comprises more than one chemical moiety 103 (e.g., a linker group comprises 1,2,3, 4,5, or 6 chemical moieties 103).

In embodiments, the linker group comprises: one chemical moiety 101, one spacer moiety 102, and one chemical moiety 103. In embodiments, the linker group consists of: one chemical moiety 101, one spacer moiety 102, and one chemical moiety 103.

In embodiments, the linker group has the structure of sub-structure (I):

-101-102-103-，(I)

wherein

"101" represents a chemical moiety 101;

"102" represents a spacer moiety 102; and is

"103" represents a chemical moiety 103.

In embodiments, the chemical moiety 101 is capable of forming a covalent bond with a surface of a microorganism.

In embodiments, chemical moiety 101 is capable of forming a covalent bond with the surface of a microorganism in the presence of one or more agents that facilitate coupling (also referred to herein as coupling agents).

In embodiments, the agent that facilitates coupling comprises glutaraldehyde, formaldehyde, paraformaldehyde, 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide (EDC), N '-Dicyclohexylcarbodiimide (DCC), N-cyclohexyl-N' - (2-morpholinoethyl) carbodiimide-methyl-p-toluenesulfonate (CMC), diisopropylcarbodiimide (DIC), (1- [ bis (dimethylamino) methylene ] carbodiimide)]-1H-1,2,3-triazolo [4,5-b]Pyridinium 3-oxide Hexafluorophosphate) (HATU), woltend reagent,N,N' -carbonyldiimidazole, N-hydroxysuccinimide (NHS) or N-hydroxysulfosuccinimide (sulfo-NHS) or any combination thereof.

In embodiments, the agent that facilitates coupling includes aldehydes, acrylates, amides, imides, anhydrides, chlorotriazines, epoxides, isocyanates, isothiocyanates, organic acids, monomers, polymers, silanes, or silicates (silcates), or any combination thereof.

In embodiments, the agent that facilitates coupling comprises a carbodiimide, phosphonium salt, or ammonium salt, or any combination thereof.

In embodiments, the agent that facilitates coupling comprises glutaraldehyde, N- (3-dimethylaminopropyl) -N' -Ethylcarbonate (EDC), (1- [ bis (dimethylamino) methylene)]-1H-1,2,3-triazolo [4,5-b]Pyridinium 3-oxideCompound Hexafluorophosphate) (HATU), (O-benzotriazol-1-yl-N, N, N, N-tetramethyluronium Hexafluorophosphate) (HBTU),N-hydroxysuccinimide (NHS),N,N'-Dicyclohexylcarbodiimide (DCC), diisopropylcarbodiimide (DIC), hydroxy-3,4-dihydro-4-oxo-1,2,3-benzotriazine (HOOBt), hydroxybenzotriazole (HOBT), 1-hydroxy-7-azabenzotriazole (HOAt), (N- (3-dimethylaminopropyl) -N' -Ethylcarbodiimide (EDAC), 4- (N, N-dimethylamino) pyridine (DMAP), benzotriazol-1-yloxy-tris (dimethylamino) -phosphonium hexafluorophosphate (BOP), benzotriazol-1-yloxy-tripyrrolidino-phosphonium hexafluorophosphate (PyBOP), bromo-tripyrrolidino-phosphonium hexafluorophosphate (PyBrOP), 7-aza-benzotriazol-1-yloxy-tripyrrolidino phosphonium hexafluorophosphate (PyAOP), ethylcyano (hydroxyimino) acetate-O2) -tris- (1-pyrrolidinyl) -phosphonium hexafluorophosphate (PyOxim), 3- (diethoxy-yloxy) -benzo [ xft 3763-xl ] 3763 zft 37 [ hod ] 37 ] x]Triazin-4 (3H) -one (DEPBT), 2- (6-chloro-1H-benzotriazol-1-yl) -N, N, N ', N' -tetramethylammonium Hexafluorophosphate (HCTU), N- [ (5-chloro-1H-benzotriazol-1-yl) -dimethylamino-morpholino]-uronium hexafluorophosphate N-oxide (HDMC), 1- [1- (cyano-2-ethoxy-2-oxoethylene (ethylidene) aminooxy) -dimethylamino-morpholino]-uronium hexafluorophosphate (COMU), 2- (1-oxy-pyridin-2-yl) -1,1,3,3-tetramethylisothiouronium tetrafluoroborate (TOTT), tetramethylfluoroamidinium hexafluorophosphate (TFFH), N-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline (EEDQ), 2-propanephosphonic anhydride (PPA), triphosgene, 1,1' -Carbonyldiimidazole (CDI), [ (6-nitrobenzotriazol-1-yl) oxy]Tris (pyrrolidino) phosphonium hexafluorophosphate (PyNOP), [ [6- (trifluoromethyl) benzotriazol-1-yl]Oxy radical]Tris (pyrrolidino) phosphonium hexafluorophosphate (PyFOP), [ [ 4-nitro-6- (trifluoromethyl) benzotriazol-1-yl]Oxy radical]Tris (pyrrolidino) phosphonium hexafluorophosphate (PyNFOP), [ (6-nitrobenz-triazol-1-yl) oxy]Tris (dimethyl-amino) phosphonium hexafluorophosphates (NOP), 1-β-naphthalenesulfonyloxybenzotriazoles (NSBt), 1-. Beta. -naphthalenesulfonyloxy-6-nitrobenzotriazoles (N-NSBt), tetramethylfluoroamidinium hexafluorophosphate (TFFH), bis (tetramethylene) fluoroamidinium hexafluorophosphate (BTFFH), 1,3-dimethyl-2-fluoro-4,5-dihydro-1H-imidazolium hexafluorophosphate (DFIH), cyanuric Chloride (CC) or 2,4-dichloro-6-methoxy-1,3,5-triazine (DCMT) and 2-chloro-4,6-dimethoxy-1,3,5-triazine (CDMT) or any combination thereof.

In embodiments, the agent that facilitates coupling comprises EDC, HATU, HBTU, NHS, DCC, HOBT, or PyBOP, or any combination thereof.

In embodiments, the agent that facilitates coupling comprises EDC, DCC, CMC, DIC, or HATU, or any combination thereof.

In embodiments, the agent that facilitates coupling comprises glutaraldehyde, formaldehyde, or paraformaldehyde, or any combination thereof.

In embodiments, chemical moiety 101 is capable of forming a non-covalent interaction with a surface of a microorganism (e.g., any of the non-covalent interactions described herein). In embodiments, the non-covalent interaction comprises: ionic interactions, ion-ion interactions, dipole-dipole interactions, ion-dipole interactions, electrostatic interactions, london dispersion, van der waals interactions, hydrogen bonding, or pi-pi interactions, or any combination thereof.

In embodiments, chemical moiety 101 is capable of forming a non-covalent interaction with a surface of a microorganism, wherein the non-covalent interaction comprises an ionic interaction, a van der waals interaction, a hydrophobic interaction, a pi-pi interaction, or a hydrogen bond, or any combination thereof.

In embodiments, the chemical moiety 101 comprises a nucleophilic functional group. In embodiments, chemical moiety 101 comprises a group formed from a nucleophilic functional group.

In embodiments, the nucleophilic functional group is: amino, amido, hydrazino, hydroxyamino, hydroxy or thio (thio). In embodiments, the nucleophilic functional group is: amino, hydrazino, hydroxyamino or thio. In embodiments, the nucleophilic functional group comprises: amino, hydrazino, hydroxyamino, hydroxy or thio. In embodiments, the nucleophilic functional group is a carboxamide, N-hydroxycarboxamide, carboxyhydrazide, or guanidino group.

In embodiments, the nucleophilic functional group is-NH ₂ 、-NHNH ₂ 、-CONHOH、-CONHNH ₂ 、–ONH ₂ -OH or-SH. In embodiments, the nucleophilic functional group is-NH ₂ 、-NHNH ₂ 、-CONHNH ₂ or-ONH ₂ 。

In embodiments, chemical moiety 101 comprises an electrophilic functional group.

In embodiments, chemical moiety 101 comprises a group formed from an electrophilic functional group.

In embodiments, the electrophilic functional group comprises an aldehyde, a ketone, a carboxylic acid ester, a carboxylic acid halide (e.g., acetyl chloride), or a carboxylic acid anhydride (e.g., acetic anhydride).

In embodiments, the electrophilic functional group comprises an aldehyde, an α -haloketone, a maleimide, a succinimide, a hydroxysuccinimide, an isothiocyanate, an isocyanate, an acyl azide, a sulfonyl chloride, a tosylate, a glyoxal, an epoxide, an oxirane, a carbonate, an imido ester, an anhydride, a fluorophenyl ester, a hydroxymethylphosphine derivative, a carbonate, a haloacetyl, a chlorotriazine, a haloacetyl, an alkyl halide, an aziridine, or an acryloyl derivative. In embodiments, the electrophilic functional group is an aldehyde, an α -haloketone, a maleimide, a succinimide, a hydroxysuccinimide, an isothiocyanate, an isocyanate, an acyl azide, a sulfonyl chloride, a tosylate, a glyoxal, an epoxide, an oxirane, a carbonate, an imidoester, an anhydride, a fluorophenyl ester, a hydroxymethylphosphine derivative, a carbonate, a haloacetyl, a chlorotriazine, a haloacetyl, an alkyl halide, an aziridine, or an acryloyl derivative.

In embodiments, the electrophilic functional group comprises an aldehyde, an alpha-haloketone, a maleimide, a succinimide, or a hydroxysuccinimide group.

In embodiments, the electrophilic functional group comprises-CHO, -C (O) CH ₂ I、

。

In embodiments, the electrophilic functional group comprises-CHO, -C (O) CH ₂ I、

。

In embodiments, chemical moiety 101 comprises a group that is an alkyl, alkenyl, alkynyl, phenyl, heteroaryl, haloalkyl, hydroxyl, carbonyl, acid halide, alkoxycarbonyl) oxy, carboxyl, haloketone, alkoxy, alkoxyol (alkaxyol) (hemiacetal or) hemiketal, dialkoxy (e.g., ketal or acetal), trialkoxy (orthogonal ether), carbamoyl, amino (amonio), imino (imino), succinamido, maleimido, hydroxysuccinamido, biotin, D-biotin, azido, azo, cyanate, isocyanato, nitroxyl, cyano, isocyano, nitrosooxy, nitro, nitroso, oxime, sulfanyl, sulfinyl, sulfonyl, sulfo, cyanato (thiocyanato), isothiocyanato, thiolyl, phosphate, or borate.

In embodiments, spacer moiety 102 is hydrophobic. In embodiments, spacer moiety 102 is hydrophilic.

In embodiments, spacer moiety 102 is peptidic (e.g., derived from a peptide bond).

In embodiments, spacer moiety 102 comprises an inorganic bond. In an embodiment, spacer moiety 102 comprises an organic bond. In embodiments, spacer moiety 102 comprises only organic bonds.

In embodiments, the spacer moiety 102 is oligomeric. In embodiments, spacer moiety 102 is multimeric. In embodiments, spacer moiety 102 comprises a methylene group (-CH) ₂ -) ethylene glycol (-CH) ₂ CH ₂ O-), iminoethylene (-CH) ₂ CH ₂ NH-), vinyl alcohol (-CH) ₂ CHOH-) _x Lactic acid (-CH) (CH) ₃ ) -C (O) -O-), acrylic acid (-CH) ₂ CH ₂ (CO ₂ H) -) methacrylic acid (-CH) ₂ C(CH ₃ )(CO ₂ H) -) or AMethacrylic acid methyl ester (-CH) ₂ C(CH ₃ )(CO ₂ CH ₃ ) -) of a plurality of segments (e.g., 1 to about 300,1 to about 200,1 to about 100,1 to about 50,1 to about 25, or 1 to about 10, or 1,2,3, 4,5, 6,7, 8,9, or 10 segments).

In embodiments, spacer moiety 102 comprises a segment that is

、

、

Or

。

In embodiments, n, m, p, and q are independently integers of 1 to about 300 (e.g., 1 to about 200,1 to about 100,1 to about 50,1 to about 25, or 1 to about 10). In embodiments, n, m, p, and q are each independently 1,2,3, 4,5, 6,7, 8,9, or 10.

In an embodiment, spacer moiety 102 comprises

. In embodiments, R' is independently hydrogen or as C ₁ -C ₁₂ Alkyl radical, C ₂ -C ₁₂ Alkenyl or C ₂ -C ₁₂ A radical of alkynyl. In embodiments, o is an integer from 1 to about 300 (e.g., from 1 to about 200,1 to about 100,1 to about 50,1 to about 25, or from 1 to about 10). In embodiments, o is independently 1,2,3, 4,5, 6,7, 8,9, or 10.

In embodiments, spacer moiety 102 comprises

. In embodiments, R is independently hydrogen or as C ₁ -C ₁₂ Alkyl radical, C ₂ -C ₁₂ Alkenyl or C ₂ -C ₁₂ A radical of alkynyl. In embodiments, r is an integer from 1 to about 300 (e.g., from 1 to about 200,1 to about 100,1 to about 50,1 to about 25, or from 1 to about 10). In embodiments, r is independently 1,2,3, 4,5, 6,7, 8,9, or 10.

In embodiments, spacer moiety 102 comprises

. In embodiments, R is independently hydrogen or as C ₁ -C ₁₂ Alkyl radical, C ₂ -C ₁₂ Alkenyl or C ₂ -C ₁₂ A radical of alkynyl. In embodiments, s is an integer from 1 to about 300 (e.g., from 1 to about 200,1 to about 100,1 to about 50,1 to about 25, or from 1 to about 10). In embodiments, s is independently 1,2,3, 4,5, 6,7, 8,9, or 10.

In embodiments, spacer moiety 102 comprises

. In embodiments, t is an integer from 1 to about 300 (e.g., from 1 to about 200,1 to about 100,1 to about 50,1 to about 25, or from 1 to about 10). In embodiments, t is independently 1,2,3, 4,5, 6,7, 8,9, or 10.

In embodiments, spacer moiety 102 comprises:

。

in embodiments, spacer moiety 102 is a polymer comprising repeating groups comprising alkyl, alkoxy, ester, acrylic, amino, hydroxyl, or acylhydrazine functional groups, or any combination thereof.

In embodiments, spacer moiety 102 is:

、

、

or

Wherein n, m, p and q are as defined herein.

In embodiments, n, m, o, p, q, r, s, or t are each independently an integer from 1 to 100, 10 to 90, 10 to 80, 10 to 70, 10 to 60, 10 to 50, 10 to 40, 10 to 30, 10 to 20, or 1 to 10.

In embodiments, chemical moiety 103 comprises a group as a nucleophilic functional group.

In embodiments, chemical moiety 103 comprises a group formed from a nucleophilic functional group.

In embodiments, the nucleophilic functional group is: amino, amido, hydrazino, hydroxyamino, hydroxy or thio. In embodiments, the nucleophilic functional group is: amino, hydrazino, hydroxyamino or thio.

In embodiments, the nucleophilic functional group comprises: amino, hydrazino, hydroxyamino, hydroxy or thio. In embodiments, the nucleophilic functional group is a carboxamide, N-hydroxycarboxamide, carboxyhydrazide, or guanidino group.

In embodiments, the nucleophilic functional group is-NH ₂ 、-NHNH ₂ 、-CONHOH、-CONHNH ₂ 、–ONH ₂ -OH or-SH. In embodiments, the nucleophilic functional group is-NH ₂ 、-NHNH ₂ 、-CONHNH ₂ or-ONH ₂ 。

In embodiments, chemical moiety 103 comprises a group that is an electrophilic functional group.

In embodiments, chemical moiety 103 comprises a group formed from an electrophilic functional group.

In embodiments, the electrophilic functional group comprises an aldehyde, a ketone, a carboxylic acid ester, a carboxylic acid halide (e.g., acetyl chloride), or a carboxylic acid anhydride (e.g., acetic anhydride).

In embodiments, the electrophilic functional group comprises an aldehyde, an α -haloketone, a maleimide, a succinimide, a hydroxysuccinimide, an isothiocyanate, an isocyanate, an acyl azide, a sulfonyl chloride, a tosylate, a glyoxal, an epoxide, an oxirane, a carbonate, an imido ester, an anhydride, a fluorophenyl ester, a hydroxymethylphosphine derivative, a carbonate, a haloacetyl, a chlorotriazine, a haloacetyl, an alkyl halide, an aziridine, or an acryloyl derivative. In embodiments, the electrophilic functional group is an aldehyde, an α -haloketone, a maleimide, a succinimide, a hydroxysuccinimide, an isothiocyanate, an isocyanate, an acyl azide, a sulfonyl chloride, a tosylate, a glyoxal, an epoxide, an oxirane, a carbonate, an imido ester, an anhydride, a fluorophenyl ester, a hydroxymethylphosphine derivative, a carbonate, a haloacetyl, a chlorotriazine, a haloacetyl, an alkyl halide, an aziridine, or an acryloyl derivative.

In embodiments, the electrophilic functional group comprises an aldehyde, an α -haloketone, a maleimide, a succinimide, or a hydroxysuccinimide group.

In embodiments, the electrophilic functional group comprises-CHO, -C (O) CH ₂ I、

。

In embodiments, the electrophilic functional group comprises-CHO, -C (O) CH ₂ I、

。

In embodiments, chemical moiety 103 comprises a chemical structure that is a carbonyl, alkenyl, alkynyl, hydroxyl, amino, thiol, maleimide, succinimide, hydroxysuccinimide, biotinyl, anhydride, chlorotriazine, epoxide, isocyanate, or isothiocyanate. In embodiments, the group that is a carbonyl, alkenyl, alkynyl, hydroxyl, amino, thiol, maleimide, succinimide, hydroxysuccinimide, biotin, anhydride, chlorotriazine, epoxide, isocyanate, or isothiocyanate is capable of forming a covalent bond with an amplifier group (e.g., amplifier group 104). In embodiments, the group that is a carbonyl, alkenyl, alkynyl, hydroxyl, amino, thiol, maleimide, succinimide, hydroxysuccinimide, biotin, anhydride, chlorotriazine, epoxide, isocyanate, or isothiocyanate is capable of forming a non-covalent interaction with an amplifier group (e.g., amplifier group 104).

In embodiments, chemical moiety 103 is formed from a chemical structure that comprises a group that is a carbonyl, alkenyl, alkynyl, hydroxyl, amino, thiol, maleimide, succinimide, hydroxysuccinimide, biotin, anhydride, chlorotriazine, epoxide, isocyanate, or isothiocyanate. In embodiments, the group that is a carbonyl, alkenyl, alkynyl, hydroxyl, amino, thiol, maleimide, succinimide, hydroxysuccinimide, biotin, anhydride, chlorotriazine, epoxide, isocyanate, or isothiocyanate has formed a covalent bond with an amplifier group (e.g., amplifier group 104). In embodiments, the group that is a carbonyl, alkenyl, alkynyl, hydroxyl, amino, thiol, maleimide, succinimide, hydroxysuccinimide, biotin, anhydride, chlorotriazine, epoxide, isocyanate, or isothiocyanate has formed a non-covalent interaction with an amplifier group (e.g., amplifier group 104).

In embodiments, chemical moiety 103 comprises a group that is a carbonyl, alkenyl, alkynyl, hydroxyl, amino, thiol, maleimide, succinimide, hydroxysuccinimide, or biotin group.

In embodiments, chemical moiety 103 comprises a carbonyl, alkenyl, alkynyl, hydroxyl, amino, thiol, maleimide, succinimide, hydroxysuccinimide, or biotinyl functional group.

In embodiments, chemical moiety 103 comprises:

。

in embodiments, chemical moiety 103 comprises a group formed from a chemical structure comprising a group that is a carbonyl, alkenyl, alkynyl, hydroxyl, amino, thiol, maleimide, succinimide, hydroxysuccinimide, or biotinyl functional group.

In embodiments, linker group L has the structure of sub-structure (II):

wherein

X represents a chemical moiety 101 (e.g., any chemical moiety 101 as described herein);

r represents a spacer moiety 102 (e.g., any spacer moiety 102 as described herein);

y represents a chemical moiety 103 (e.g., any chemical moiety 103 as described herein); and is

j and k are each independently an integer from 0 to 100.

In embodiments, X is

。

In embodiments, R is

Wherein n, m, o, p, q, r, s, or t are each as described herein (e.g., an integer from 1 to about 300).

In embodiments, Y is

。

In embodiments, X is capable of forming a covalent bond with the surface of a microorganism. In embodiments, X forms a covalent bond with the surface of the microorganism.

In embodiments, X is capable of forming one or more non-covalent interactions with the surface of a microorganism. In embodiments, X forms one or more non-covalent interactions with the surface of the microorganism.

In embodiments, Y is capable of forming a covalent bond with an amplifier group 104 (e.g., a chemical or biochemical amplifier). In embodiments, Y forms a covalent bond with an amplifier group (such as amplifier group 104) (e.g., a chemical or biochemical amplifier).

In embodiments, Y is capable of forming one or more non-covalent interactions with the amplifier group 104 (e.g., a chemical or biochemical amplifier). In embodiments, Y forms one or more non-covalent interactions with an amplifier group (such as amplifier group 104) (e.g., a chemical or biochemical amplifier).

In embodiments, the linker group L is:

WGA-biotin, polymyxin B-biotin, monoclonal antibodies, polyclonal antibodies, biotinylated monoclonal antibodies, biotinylated polyclonal antibodies, europium chelate-antibodies, horseradish peroxidase-conjugated antibodies and antibody variants (e.g., fab: fragment, antigen binding (one arm); F (ab ') 2: fragment, antigen binding, including hinge region (two arms); fab' fragment, antigen binding, including hinge region (one arm); scFv: single chain variable fragment; di-scFv: dimeric single chain variable fragment; sdAb: single domain antibody; bispecific monoclonal antibody; trifunctional antibody; and BiTE: bispecific T-cell engager).

Exemplary amplifier groups include those described, for example, in International publication No. WO 2016/015027 and in International application No. PCT/US 16/42589 (each of which is incorporated by reference in its entirety).

In embodiments, the amplifier group (e.g., amplifier group 104) comprises a catalyst, a fluorophore, or a colorimetric dye. In embodiments, the amplifier group (e.g., amplifier group 104) is a catalyst, fluorophore, or colorimetric dye.

In embodiments, the amplifier group (e.g., amplifier group 104) comprises an enzyme, a catalyst, or a nanoparticle. In embodiments, the amplifier group (e.g., amplifier group 104) is an enzyme, a catalyst, or a nanoparticle.

In embodiments, the chemical amplifier group comprises a catalyst, a fluorophore, a nanoparticle, or a colorimetric dye. In embodiments, the chemical amplifier group is a catalyst, a fluorophore, a nanoparticle, or a colorimetric dye.

In embodiments, the amplifier group (e.g., amplifier group 104) comprises a catalyst. In embodiments, the amplifier group (e.g., amplifier group 104) is a catalyst.

In embodiments, the amplifier group (e.g., amplifier group 104) comprises a fluorophore. In embodiments, the amplifier group (e.g., amplifier group 104) is a fluorophore. Exemplary fluorophores include those described in table 1 of international application No. PCT/US 16/42589 (which is incorporated by reference in its entirety).

In embodiments, the amplifier group (e.g., amplifier group 104) comprises a colorimetric dye. In embodiments, the amplifier group (e.g., amplifier group 104) is a colorimetric dye.

In embodiments, the amplifier group (e.g., amplifier group 104) comprises an enzyme. In embodiments, the amplifier group (e.g., amplifier group 104) is an enzyme.

In embodiments, the amplifier groups (e.g., amplifier groups 104) comprise nanoparticles. In embodiments, the amplifier groups (e.g., amplifier groups 104) are nanoparticles.

In embodiments, the amplifier group (e.g., amplifier group 104) comprises a lanthanide.

In embodiments, the amplifier group (e.g., amplifier group 104) comprises a lanthanide that is europium, strontium, terbium, samarium, or dysprosium. In embodiments, the amplifier group (e.g., amplifier group 104) comprises a lanthanide selected from: europium, strontium, terbium, samarium and dysprosium.

In embodiments, the amplifier group (e.g., amplifier group 104) comprises an organic fluorophore.

In embodiments, the amplifier group (e.g., amplifier group 104) comprises a fluorophore as a coordination complex.

In embodiments, the amplifier group (e.g., amplifier group 104) comprises a europium coordination complex. In embodiments, the coordination complex is a europium coordination complex. In embodiments, the amplifier group (e.g., amplifier group 104) comprises a ruthenium coordination complex. In embodiments, the coordination complex is a ruthenium coordination complex. In embodiments, the amplifier group (e.g., amplifier group 104) comprises a rhenium coordination complex. In embodiments, the coordination complex is a rhenium coordination complex. In embodiments, the amplifier group (e.g., amplifier group 104) comprises a palladium coordination complex. In embodiments, the coordination complex is a palladium coordination complex. In embodiments, the amplifier group (e.g., amplifier group 104) comprises a platinum coordination complex. In embodiments, the coordination complex is a platinum coordination complex.

In embodiments, the amplifier group (e.g., amplifier group 104) comprises a chemiluminescent group, a quantum dot, an enzyme, an iron coordination catalyst, a europium coordination complex, a ruthenium coordination complex, a rhenium coordination complex, a palladium coordination complex, a platinum coordination complex, a samarium coordination complex, a terbium coordination complex, or a dysprosium coordination complex.

In embodiments, the amplifier group (e.g., amplifier group 104) comprises a chemiluminescent group. In an embodiment, the amplifier groups (e.g., amplifier groups 104) comprise quantum dots. In embodiments, the amplifier group (e.g., amplifier group 104) comprises an enzyme. In embodiments, the amplifier group (e.g., amplifier group 104) comprises an iron coordination catalyst. In embodiments, the amplifier group (e.g., amplifier group 104) comprises a europium coordination complex. In embodiments, the amplifier group (e.g., amplifier group 104) comprises a ruthenium coordination complex. In embodiments, the amplifier group (e.g., amplifier group 104) comprises a rhenium coordination complex. In embodiments, the amplifier group (e.g., amplifier group 104) comprises a palladium coordination complex. In embodiments, the amplifier group (e.g., amplifier group 104) comprises a platinum coordination complex. In embodiments, the amplifier group (e.g., amplifier group 104) comprises a samarium coordination complex. In embodiments, the amplifier group (e.g., amplifier group 104) comprises a terbium coordination complex. In an embodiment, the amplifier radical (e.g., amplifier radical 104) comprises a dysprosium coordination complex.

In embodiments, the amplifier group 104 comprises a moiety that is:

。

in embodiments, the amplifier group 104 comprises a moiety that is:

。

in embodiments, the amplifier group 104 is a catalyst or an enzyme. In embodiments, the amplifier group is horseradish peroxidase, alkaline phosphatase, acetylcholinesterase, glucose oxidase, beta-D-galactosidase, or beta-lactamase.

In embodiments, the amplifier group 104 is horseradish peroxidase.

In embodiments, the amplifier group 104 is a fluorophore or a colorimetric dye.

Suitable fluorophores and colorimetric dyes are well known to those skilled in the art and are described inThe Molecular Probes® Handbook: A Guide to Fluorescent Probes and Labeling Technologies11 th edition (2010) and Gomes, fernandes and LimaJ. Biochem. Biophys. Methods65 (2005) pp 45-80, which is incorporated herein by reference in its entirety. Exemplary fluorophores also include those described in, for example, international publication No. WO 2016/015027 and International application No. PCT/US 16/42589 (each of which is incorporated by reference in its entirety).

Examples of suitable fluorophores or colorimetric dyes include, but are not limited TO, ethidium bromide, propidium iodide, SYTOX green, phenanthridines, acridines, indoles, imidazoles, cyanines, TOTO, TO-PRO, SYTO, 5-carboxy-2,7-dichlorofluorescein, 5-carboxyfluorescein (5-FAM), 5-carboxynaphthyl fluorescein, 5-carboxytetramethylrhodamine (5-TAMRA), 5-FAM (5-carboxyfluorescein), 5-HAT (hydroxytryptamine), 5-ROX (carboxy-X-rhodamine), 6-carboxyrhodamine 6G, 7-amino-4-methylcoumarin, coumarin, and the like 7-Aminoactinomycin D (7-AAD), 7-hydroxy-4-methylcoumarin, 9-amino-6-chloro-2-methoxyacridine, ACMA (9-amino-6-chloro-2-methoxyacridine), acridines, alexa Fluors, alizarin, allophycocyanin (APC), AMCA (aminomethylcoumarin), bodipy, carboxy-X-rhodamine, catecholamine, fluorescein (FITC), hydroxycoumarin, lissamine rhodamine, monobromobimane, oregon green, phycoerythrin, SYTO, thiodicarbocyanine (DiSC 3), thioflavin, X-rhodamine, C or tetramethylrhodamine isothiocyanate.

In embodiments, the amplifier group 104 is an organometallic compound, a transition metal complex, or a coordination complex. <xnotran> , EP 0 180 492, EP 0 321 353, EP 0 539 435, EP 0 539 477, EP 0 569 496, EP, EP, US, US, US, US, US, US, US, US, US, US, US, US, US, US, US, US, US, US, US, US, US, US, US, US, US, US, US, US, US, US, US, US, US, US, US, US, US, US, US, US, US, US, US, US, US, US, US, US, US, US, US, US, US, US, US, US, US, US, US, US, US, US, US US, . </xnotran> Exemplary organometallic compounds, transition metal complexes, or coordination complexes also include those described, for example, in international publication No. WO 2016/015027 and international application No. PCT/US 16/42589 (each of which is incorporated by reference in its entirety).

In embodiments, the amplifier group 104 is a lanthanide coordination complex.

In embodiments, the lanthanide coordination complex is a complex between a lanthanide (e.g., eu or Tb) and a tetradentate ligand.

In embodiments, the lanthanide coordination complex is a complex between a lanthanide (e.g., eu or Tb) and a cryptate ligand.

In embodiments, the amplifier group 104 is a coordination complex of lanthanum (La), cerium (Ce), praseodymium (Pr), neodymium (Pm), samarium (Sm), europium (Eu), gadolinium (Gd), terbium (Tb), dysprosium (Dy), holmium (Ho), erbium (Er), thulium (Tm), ytterbium (Yb), lutetium (Lu), ruthenium (Ru), rhodium (Rh), palladium (Pd), osmium (Os), iridium (Ir), or platinum (Pt).

In an embodiment, amplifier group 104 is a coordination complex of rare earth metals, which collectively refers to 17 elements (the lanthanide elements) consisting of the group of 15 elements from lanthanum, atomic number 57, to lutetium, atomic number 71, and two additional elements consisting of scandium, atomic number 21, and yttrium, atomic number 39. Specific examples of the rare earth metal include europium, terbium, lanthanum, cerium, praseodymium, neodymium, promethium, samarium, gadolinium, dysprosium, holmium, erbium, thulium, ytterbium, lutetium, scandium, and yttrium, preferably europium and terbium, and more preferably europium.

In embodiments, the amplifier group 104 is a coordination complex of a lanthanide (e.g., europium or terbium) with diethylene triamine tetraacetic acid or a cryptate ligand.

In embodiments, the amplifier group 104 is a coordination complex of a lanthanide (e.g., europium or terbium) with diethylenetriamine tetraacetic acid.

In embodiments, the amplifier group 104 is a coordination complex of a lanthanide (e.g., europium or terbium) with a cryptate ligand.

In embodiments, the signaling agent (e.g., chemical signaling agent) comprises or is formed from:

eu-cryptate-maleimide

Eu-cryptate-NHS

Eu-cryptate-diamine

Eu-N1-ITC (Delfia)

Eu-N1-DTA

Eu-N1-amino group

Eu-N1-iodoacetamido group

。

In embodiments, the signaling agent may comprise one or more paramagnetic metal chelates to form a contrast agent. Preferred paramagnetic metal ions have an atomic number of 21-29, 42, 44 or 57-83. This includes ions of the transition metal or lanthanide series that have one, and more preferably five or more unpaired electrons and a magnetic moment of at least 1.7 bohr magnetons. Preferred paramagnetic metals include chromium (III), manganese (II), manganese (III), iron (II), iron (III), cobalt (II), nickel (II), copper (II), praseodymium (III), neodymium (III), samarium (III), gadolinium (III), terbium (III), dysprosium (III), holmium (III), erbium (III), europium (III) and ytterbium (III). In addition, the signaling agents of the present invention may further comprise one or more superparamagnetic particles:

in embodiments, the signaling agent may comprise one or more metals included in the metal complex with or as part of the fluorescent compound: the metal complex includes compounds having Al, zn, be, etc.; rare earth metals such as Tb, eu or Dy; or a transition metal such as Pt or Ir as a central metal, and a metal complex having an oxadiazole, thiadiazole, phenylpyridine, phenylbenzimidazole, or quinoline structure as a ligand, such as an aluminum quinolinol complex, a benzoquinolinol beryllium complex, a benzoxazole zinc complex, a benzothiazole zinc complex, an azomethyl zinc complex, a porphyrin zinc complex, and a europium complex.

In embodiments, the signaling agent may comprise a luminophore (donor) characterized by high luminescence quantum efficiency and long luminescence decay time (> 100 ns). Preferred luminophores are cations, metal-organic complexes of palladium, rhodium, platinum, ruthenium, osmium, rare earths, in particular europium and lanthanum. The organic part of these metal-organic complexes may consist of ligands from the group of porphyrins, bipyridines, phenanthrolines or other heterocyclic compounds, for example.

In embodiments, the signaling agent capable of binding to the surface of the microorganism comprises an antibody (e.g., monoclonal or polyclonal), a modified antibody (e.g., biotinylated monoclonalMonoclonal antibodies, biotinylated polyclonal antibodies, europium chelate-antibodies, horseradish peroxidase-conjugated antibodies), antibody variants (e.g., fab: fragment, antigen binding (one arm); f (ab') ₂ : fragments, antigen binding, including the hinge region (two arms); fab': fragments, antigen binding, including the hinge region (one arm); scFv: a single-stranded variable fragment; a di-scFv: a dimeric single-stranded variable fragment; sdAb: a single domain antibody; a bispecific monoclonal antibody; a trifunctional antibody; and BiTE: bispecific T cell engagers), WGA-biotin, polymyxin B-biotin, lectins, natural peptides, synthetic and/or natural ligands, synthetic and/or natural polymers, synthetic and/or natural glycopolymers, carbohydrate-binding proteins and/or polymers, glycoprotein-binding proteins and/or polymers, charged small molecules, other proteins, bacteriophages and/or aptamers.

The term "antibody" as used herein refers to any antibody, modified antibody or antibody fragment of the type described herein or of the type as known in the art. Thus, "antibody-containing signaling agents" include, for example, signaling agents comprising unmodified monoclonal antibodies, fab fragments, and trifunctional antibodies.

In embodiments, the signaling agent capable of binding to the surface of the microorganism comprises a lanthanide coordination complex, biotin, an antibody, and/or an enzyme.

In embodiments, the signaling agent capable of binding to a microbial surface comprises or is formed from a structure comprising an antibody, lectin, natural peptide, synthetic and/or natural ligand, synthetic and/or natural polymer, synthetic and/or natural glycopolymer, carbohydrate-binding protein and/or polymer, glycoprotein-binding protein and/or polymer, charged small molecule, other protein, bacteriophage, and/or aptamer.

In embodiments, the signaling agent capable of binding to the microbial surface comprises an amplifier group 104 comprising a lanthanide coordination complex, and/or an enzyme and streptavidin and/or an antibody and/or an aptamer.

In embodiments, the signaling agent capable of binding to a microbial surface comprises a binding moiety comprising a polyclonal and/or monoclonal antibody.

In embodiments, the signaling agent capable of binding to a microbial surface comprises a binding moiety comprising a modified antibody. Exemplary modified antibodies include biotinylated monoclonal antibodies, biotinylated polyclonal antibodies, europium chelate-antibodies, and horseradish peroxidase conjugated antibodies.

In embodiments, the signaling agent capable of binding to a microbial surface comprises a binding moiety comprising an antibody variant. Exemplary antibody variants include Fab: fragment, antigen binding (one arm); f (ab') ₂ : fragments, antigen binding, including the hinge region (two arms); fab': fragments, antigen binding, including the hinge region (one arm); scFv: a single-stranded variable fragment; a di-scFv: a dimeric single-stranded variable fragment; sdAb: a single domain antibody; a bispecific monoclonal antibody; a trifunctional antibody; and BiTE: bispecific T-cell engagers).

In embodiments, the signaling agent capable of binding to the surface of the microorganism comprises WGA-biotin or polymyxin B-biotin.

In embodiments, the signaling agent capable of binding to a microbial surface comprises a binding moiety comprising a synthetic and/or natural ligand and/or peptide.

In embodiments, the ligand and/or peptide is selected from the group consisting of bis (zinc-lutidine), TAT peptide, serine protease, cathelicidins, cationic dextrins, cationic cyclodextrins, salicylic acid, lysine, and combinations thereof.

In embodiments, the signaling agent capable of binding to a microbial surface comprises a binding moiety comprising a synthetic and/or natural polymer and/or a carbohydrate polymer.

In an embodiment of the present invention, the substrate is, the natural and/or synthetic polymers are linear or branched and are selected from the group consisting of amylopectin poly (A), (B), (C)N- [3- (dimethylamino) propyl group]Methacrylamide), poly (ethyleneimine), poly-L-lysine, poly [ 2- (meth) acrylic acid [ ((meth) acrylic acid)N, N-dimethylamino) ethyl ester]And combinations thereof.

In embodiments, the natural and/or synthetic polymers and/or sugar polymers comprise moieties including, but not limited to, chitosan, gelatin, dextran, trehalose, cellulose, mannose, cationic dextrans and cyclodextrins, quaternary amines, pyridinium tribromide, histidine, lysine, cysteine, arginine, sulfonium, phosphonium, or combinations thereof, including, but not limited to, co-block, graft, and alternating polymers.

In embodiments, the signaling agent capable of binding to a microbial surface comprises a binding moiety comprising a glycoprotein selected from the group consisting of mannose-binding lectins, other lectins, annexins, and combinations thereof.

In embodiments, a signaling agent capable of binding to a microbial surface comprises:

an antibody; and

a europium coordination complex.

In embodiments, the signaling agent capable of binding to the surface of the microorganism comprises a linker group L comprising NH ₂ -PEG-Biotin (2K), NH ₂ -PEG-biotin (4K), sulfo-NHS-biotin, WGA-biotin or polymyxin B-biotin.

In embodiments, a signaling agent capable of binding to a microbial surface comprises a europium complex comprising:

。

in embodiments, a signaling agent capable of binding to a microbial surface comprises a europium complex comprising:

。

as disclosed in the working examples below and throughout the specification and drawings, the present invention provides at least:

for 75 strains of 12 bacterial species (including β -lactams with gram-negative rods) >89.9% MIC identity (± 1 dilution) between the presently disclosed method and CLSI standard, with no major/very large errors;

equivalent MIC between the presently disclosed method directly from positive blood culture and CLSI Standard blood culture sample treatment

Detection as low as 2X 10 ³ CFU/ml of gram positive and negative species;

non-specific binding of a microorganism by a signaling agent;

using a europium preparation;

semi-automatic device with data output.

Additional teachings related to the present invention are described in one or more of the following: EP139675; EP64484; US 2013/0217063; US 2014/0278136; US 2014/0323340; US 2014/0363817; US 2015/0064703; US 2015/0337351; US 2016/0010138; US 3,798,320; US 4,565,790; US 4,647,536; US 4,808,541; US 4,927,923; US 5,457,185; US 5,489,401; US 5,512,493; US 5,527,684; US 5,627,074; US 5,665,554; US 5,695,946; US 6,284,470; US 6,385,272; US 6,844,028; US 7,341,841; US 7,629,029; US 7,868,144; US 8,178,602; US 8,895,255; PCT/US2016/042589; and WO/2016015027, each of which is incorporated herein by reference in its entirety.

Any of the above aspects and embodiments may be combined with any other aspects or embodiments as disclosed in the figures, summary and/or detailed description (including the examples below).

Although methods and materials similar or equivalent to those described herein can also be used in the practice or testing of the present invention, suitable methods and materials are described below. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. Citation of references herein is not an admission that it is prior art with respect to the claimed invention. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting.

Examples

Example 1: the present invention provides a rapid and accurate determination of the Minimum Inhibitory Concentration (MIC) of an antimicrobial agent

In this example, the assay will be used for rapid assays against Staphylococcus aureus: (Staphylococcus aureus) Or Pseudomonas aeruginosaPseudomonas aeruginosa) The invention of Minimum Inhibitory Concentration (MIC) of antimicrobial agent of (1) with Staphylococcus aureus requiring overnight incubation: (S. aureus) Or Pseudomonas aeruginosaP. aeruginosa) Was compared with the standard method of (1).

Staphylococcus aureus (S.aureus) was incubated at 37 ℃ with vigorous shaking using Mueller-Hinton (MH) liquid mediumStaphylococcus aureus) The culture (ATCC strain 29213) was grown overnight. Meanwhile, two sterile 96-well microplates were prepared with serial dilutions of clindamycin ranging from 32. Mu.g/ml to 0.125. Mu.g/ml and a clindamycin-free control (both in MH liquid medium). Staphylococcus aureus from overnight cultures was then transferred using McFarland standard techniques (S. aureus) The concentration is set to 5X 10 ⁵ CFU/ml, for optical density readings at 600 nm. A microplate (each well containing 200 μ L) was inoculated with the prepared dilution of the antimicrobial agent, and incubated at 37 ℃ for 3.5 hours for determination of antimicrobial sensitivity using the invention disclosed herein (e.g., "Rapid-AST" technology) or overnight at 37: (>12 hours) for OD ₆₀₀ And (6) comparison. After 4 hours, the quick-AST microplate is removed from the shaking incubator, and the polyclonal rabbit is used for resisting staphylococcus aureus (1)S. aureus) Horseradish peroxidase (HRP) conjugate of the antibody (Fitzgerald Industries International, inc.) was added to each well. The plates were then shaken at room temperature for 20 minutes to allow them to bind, and the microplates were then prepared in 4,000 formatgCentrifugation to pellet the remaining intact bacteria. Then the MH liquid medium was aspirated and sterile liquid medium was added, and washed 3 times in total. After the final aspiration, add3,3',5,5' -Tetramethylbenzidine (TMB) and hydrogen peroxide in the presence of a stabilized chromogenic solution (ThermoFisher) using a microplate reader (microplate reader) ((TM))VmaxMolecular Devices) was monitored for 10 minutes at 650nm and 450 nm. After overnight incubation, the OD was removed from the incubator ₆₀₀ Control microplate, and direct reading of optical density at 600nm ((II))VmaxMolecular Devices). Finally, the MIC was determined for each CLSI standard from the data, as shown in fig. 5. The MICs determined by both techniques were identical: 0.125 Microgram/ml.

Similarly, pseudomonas aeruginosa was subjected to vigorous shaking using MH liquid medium at 37 ℃Pseudomonas aeruginosa) The culture of (ATCC strain 27853) was grown overnight. At the same time, two sterile 96-well microplates were prepared with serial dilutions of ceftazidime from 32 μ g/ml to 0.125 μ g/ml and no ceftazidime control (both in MH liquid medium). Pseudomonas aeruginosa from overnight cultures was then treated using McFarland standard techniques (P. aeruginosa) The concentration is set to 5X 10 ⁵ CFU/ml, for optical density readings at 600 nm. These microplates (each well containing 200 μ L) were inoculated with the prepared antimicrobial dilution and incubated at 37 ℃ for 3.5 hours for determination of antimicrobial sensitivity using the invention disclosed herein or overnight at 37 ℃ (S) ((S))>12 hours) for OD ₆₀₀ And (6) comparison. After 4 hours, the "quick-AST" microplates were removed from the shaking incubator and the polyclonal rabbit anti-Pseudomonas aeruginosa (Pseudomonas aeruginosa)P. aeruginosa) A solution of horseradish peroxidase (HRP) conjugate of antibody (Abcam) was added to each well. The plates were then shaken at room temperature for 20 minutes to allow them to bind, and the microplates were then prepared in 4,000 formatgCentrifugation to pellet the remaining intact bacteria. Then the MH liquid medium was aspirated and sterile liquid medium was added, and washed 3 times in total. After the final aspiration, a stabilized chromogenic solution (ThermoFisher) consisting of 3,3',5,5' -TMB and hydrogen peroxide was added and applied to a microplate reader (R) ((R))VmaxMolecular Devices) were monitored for 10 minutes at 650nm and 450 nm. The data shown in fig. 6 depicts the 5 minute point from the beginning of incubation with the detection solution. After overnight incubation, the OD was removed from the incubator ₆₀₀ Control microplate, and direct reading of optical Density at 600 nm: (VmaxMolecular Devices). Finally, the MIC was determined for each CLSI standard from the data, as shown in fig. 6. The MICs determined by both techniques were identical: 4. mu g/ml.

The accuracy of the invention in determining the MIC clearly shows that the slope of the downward region of the data (in fig. 5 and in fig. 6) is nearly identical between the invention and the overnight culture.

These data show that the present invention is capable of accurately determining the MIC of an antimicrobial agent, which is at least as accurate as the standard method that requires overnight incubation of a bacterial culture.

Example 2: the present invention provides a rapid and accurate determination of the Minimum Inhibitory Concentration (MIC) of an antimicrobial agent for antimicrobial resistant bacteria

In this example, the present invention was used to compare antimicrobial resistant Pseudomonas aeruginosa (P.aeruginosa)P. aeruginosa) Staphylococcus aureus (1)S. aureus) And Escherichia coli (E. coli) MIC of the Strain, antimicrobial sensitive Pseudomonas aeruginosaP. aeruginosa) Staphylococcus aureus (1)S. aureus) And Escherichia coli (E. coli) MIC of the strain.

Sensitive P.aeruginosa was assayed as described in example 1P. aeruginosa) The MIC of strain ATCC 27853, the key difference being the use of imipenem as an antimicrobial agent (serial dilutions of 32 μ g/ml to 0.125 μ g/ml were used). Resistant P.aeruginosa was similarly determined (seeP. aeruginosa) MIC of strain ATCC BAA-2108. The same 96-well microplate was used for both strains, 48 wells dedicated to each strain. The experiment was repeated 3 times with similar results and the resulting data are shown in figure 7. The MIC of the sensitive strain is 2 mug/ml; the MIC of the resistant strain was 32. Mu.g/ml.

For Staphylococcus aureus (S.) (S. aureus) The same procedure as described in example 1 above was used, except that methicillin was used as the antimicrobial agent and resistant strain ATCC 43300 was used. For Escherichia coli (E.coli) (II)E. coli) Use of the composition of the invention in the treatment of Pseudomonas aeruginosaP. aeruginosa) In the same manner as described above, the procedure is, except that Escherichia coli (A), (B), (C)E. coli) Sensitive (25922) and resistant (35218) strains and use ampicillin as an antimicrobial agent and polyclonal rabbit anti-E.coli (E.coli)E. coli) The HRP conjugate of the antibody (Abcam) acts as a chemical moiety that allows the signaling agent to bind to the bacteria. (ii) the ` Rapid-AST ` value and OD after incubation for 5 min with detection solution ₆₀₀ The overnight controls were compared and the data compiled in figure 8.

These data show that the present invention is able to accurately distinguish the MIC of an antimicrobial agent for a resistant bacterial strain of the antimicrobial agent from the MIC of the same bacterial strain to which the antimicrobial agent is sensitive.

Example 3: the present invention provides detectable signals at microbial concentrations two hundred times lower than those required by standard methods

In this example, the concentration of the microorganism required to provide a detectable signal in the present invention is compared to the concentration of Staphylococcus aureus which requires overnight incubation (S.aureus)S. aureus) The standard method of (3) for comparison.

Staphylococcus aureus (S.aureus) as described in example 1S. aureus) The culture was carried out overnight. Serial dilutions of overnight colonies were performed in 96-well microplates and absorbance read at 600 nm. These values were compared to a McFarland standard to obtain the bacterial concentration in CFU/ml. The quantifiable region of the curve is shown in FIG. 9 (OD) ₆₀₀ ) (ii) a The experiment was repeated three times with similar results. Staphylococcus aureus (Staphylococcus aureus) (see (1) in example 2)S. aureus) Specific signaling agent Staphylococcus aureus (S.) (S. aureus) For 20 minutes in a similar dilution series. Following the procedure of example 2, the microorganisms were centrifuged and washed three times and a detection solution was added.

In FIG. 9, the resulting absorbance vs. McFarland standard determination of Staphylococcus aureus is shown (S.aureus)S. aureus) And (4) concentration. Starting bacterial concentration from the Clinical Laboratory Standards Institute (CLSI) -Standard AST experiment (i.e., 5X 10) as indicated by the arrow ⁵ CFU/ml) a "quick-AST" signal is visible. In contrast, optical signals are up to-10 ⁸ CFU/ml can achieve accurate quantification.

These data show that the present invention is capable of providing a detectable and useful signal at microbial culture concentrations that are two hundred fold lower than those required by standard methods.

Example 4: the present invention provides MIC values across species and strains of a variety of pathogenic bacteria similar to those obtained from the CLSI reference method, but for a time significantly shorter than that required for the CLSI method.

In this example, the present invention for rapidly determining the MIC of an antimicrobial agent for a variety of pathogenic bacteria is compared to the Clinical Laboratory Standard Institute (CLSI) method.

As shown in fig. 10, MIC determinations were obtained for six bacteria after 3.5 hours incubation, while CLSI AST reference method determinations were obtained after 16 hours incubation (for ampicillin treated cultures) or 24 hours incubation (for oxacillin treated cultures). Drugs, signaling agents/chemical moieties ("antibody-HRP conjugates") and bacterial strains are listed in fig. 11. For signaling agents/chemical moieties, wheat Germ Agglutinin (WGA) HRP conjugate was used for staphylococcus epidermidis: (S. epidermidis) The test, and the "rapid-AST" assay followed the procedure of example 2 above. All clinical isolates were unidentified samples and were sub-cultured at least twice prior to use. A total of 87 individual samples were tested, including but not limited to the following bacterial species: escherichia coli (A)E. coli) Staphylococcus aureus (1)S. aureus) Pseudomonas aeruginosaP. aeruginosa) Klebsiella pneumoniae (K.pneumoniae) ((B))K. pneumoniae) Enterococcus faecalis: (A)E. faecalis) Coagulase-Negative staphylococci (Coagula-Negative) Staphylococci) Proteus mirabilis (A), (B)P. mirabilis) Enterococcus faecium (C. Faecium)E. faecium)、E. clocaeAnd Acinetobacter baumannii: (A), (B)A. baumannii). Notably, the bacterial species tested in this example (except Proteus vulgaris: (A), (B), (C), and (C)P. mirabilis) Together responsible for many clinical laboratories>90% of positive blood cultures. Therefore, the invention has obvious clinical relevance to human infectious diseases. MIC values were highly similar between the invention and the CLSI method, but the invention required three and a half hours of incubation, whereas the CLSI methodSixteen or twenty-four hours incubation was required.

These data show that the present invention is capable of accurately determining the MIC of an antimicrobial agent, which is at least as accurate as the CLSI method, but takes significantly less time to determine the MIC; thus, the present invention greatly reduces the time before an appropriate treatment regimen (i.e., a particular antimicrobial agent and at a particular dosage) is provided to a patient.

Example 5: for Staphylococcus aureus (S.) (S. aureus) And Klebsiella pneumoniae: (K. pneumoniae) Samples, across a wide range of antimicrobial agents, the present invention provides MICs values similar to those obtained from the CLSI reference method, but in significantly less time.

In this example, when Staphylococcus aureus (S.aureus) ((S.aureus)) is treatedS. aureus) (gram-positive bacteria) or Klebsiella pneumoniaeK. pneumoniae) (gram negative bacteria) the present invention is used to rapidly determine the MIC of various antimicrobial agents and compare them to the MIC values obtained by the CLSI method.

Commercial full plate dried antimicrobial board SensiTitre (ThermoFisher) was used in the method of the invention as described in example 2 above, wherein bacterial viability was assessed at 4 hours. Representative Staphylococcus aureus bacterium (S.) (S. aureus) And Klebsiella pneumoniae: (K. pneumoniae) The results are shown in fig. 12A to 12C. For the treatment of diseases other than Staphylococcus aureus: (A)S. aureus) Erythromycin experiments performed and with Klebsiella pneumoniae (R) ()K.pneumonia) All experiments, except the tetracycline and imipenem experiments performed, showed excellent agreement between the MIC values obtained from the "fast-AST" of the invention and the results obtained from CLSI; however, the difference between the present invention and CLSI results is a "minor error" according to the FDA, see fig. 12C.

These data show that the present invention is capable of accurately determining the MIC of multiple antimicrobial agents for two different bacterial species, which is at least as accurate as the CLSI method, but takes significantly less time to determine the MIC; thus, the present invention greatly reduces the time before an appropriate treatment regimen (i.e., a particular antimicrobial agent and at a particular dosage) is provided to a patient.

Example 6: using a plurality of Staphylococcus aureus (S. aureus) And Escherichia coli (E. coli) Clinical strains, across a wide variety of antimicrobial agents, the present invention provides MICs values similar to those obtained from the CLSI reference method, but in significantly less time.

In this example, when Staphylococcus aureus (S.aureus) ((S.aureus)) is treatedS. aureus) (gram-positive bacteria) or Escherichia coli (E.coli)E. coli) (gram negative bacteria) the present invention is used to rapidly determine the MIC of various antimicrobial agents and compare them to the MIC values obtained by the CLSI method.

These experiments were carried out as in example 5 using SensiTitre ® panels (ThermoFisher). The same procedure as described in example 2 was used except that 50 μ L of inoculum was added to each well according to the thermo fisher instructions. The CLSI reference method was performed for 24 hours (oxacillin and vancomycin) and 18 hours (levofloxacin) and within 4 hours (including three and a half hour incubations) for all experiments using the present invention ("the rapid-AST" method). The results are shown in fig. 13A to 13C and fig. 14A to 14D. The dark lines in fig. 13A-13C and 14A-14D show the CLSI break points for each antimicrobial. The base protocol "EA" and the classification protocol "CA" are defined by the FDA in the class II guidelines of its automatic AST system. In addition, for Staphylococcus aureus (S.), (S. aureus) A plurality of "rapid-AST" assays and CLSI standard reference assays as described above were run over the course of one month to determine consistency of results; see fig. 15.

These data show that, when in Staphylococcus aureus: (S. aureus) And Escherichia coli (E. coli) When tested with multiple antimicrobial agents on clinical strains, the present invention ("rapid-AST" method) provides results consistent with the CLSI reference method, but takes significantly less time to determine MIC; thus, the present invention greatly reduces the time before an appropriate treatment regimen (i.e., a particular antimicrobial agent and at a particular dosage) is provided to a patient.

Example 7: the present invention provides a rapid and accurate determination of the MIC of various antimicrobial agents for antimicrobial resistant bacteria.

In this example, the present invention is used to determine the resistance of various antimicrobial agents to antimicrobial agents: (E.coli) ((II))E. coli) MIC of the Strain and antimicrobial sensitive Escherichia coli: (E. coli) MIC of the strain.

Escherichia coli (QC strain, ATCC 25922) and clinically resistant Escherichia coli (Escherichia coli)E. coli) ("clinical") both were cultured under standard sterile conditions in Mueller-Hinton (MH) liquid medium overnight at 37 ℃ with shaking. Coli from overnight cultures were then grown using McFarland standard techniques (E. coli) The concentration is set to 5X 10 ⁵ CFU/ml, for optical density readings at 600 nm. At the same time, two sterile 96-well microplates were prepared with serial dilutions of the specific antimicrobial (see below) and a no antimicrobial (saline) control (both in MH broth). Microplates (each well containing 200 μ L) were inoculated with the prepared antimicrobial dilutions and incubated at 37 ℃ for 3 hours and 45 minutes for assays using the present invention ("rapid-AST" technique). After 3 hours and 45 minutes, the "quick-AST" microplates were removed from the shaking incubator and centrifuged at 2500g for 2.5 minutes to pellet. The MH liquid medium was then aspirated and 100 μ L of water was added to each well of both microwell plates. Then, 10 μ L of chemical moiety (here, europium-cryptate formulation) was added to each well (to 20 ng/well) and 10 μ L of 5% glutaraldehyde (as a signaling agent) was added to each well. Both microplates were then shaken at 300rpm for 30 minutes. After that, both plates were centrifuged at 2500g for 2.5 minutes to pellet. The solution was aspirated and 200. Mu.L of PBS-tween wash was added to each well, followed by centrifugation to pellet. After aspirating the solution, a second identical wash of 200 μ L PBS-tween occurred, followed by final centrifugation to pellet. Time resolved fluorescence plate readings were then used on a BioTek HI plate reader. The method was carried out with the following antimicrobial agent formulation: imipenem diluted at a concentration of 8 to 0.12 μ g/ml (fig. 16); ampicillin diluted at a concentration of 32 to 0.25 mug/ml (fig. 17); ceftazidime diluted at a concentration of 32 to 0.03 mug/ml (fig. 18); based on 16 mug/ml to 0.06 mugGentamicin diluted in ml (fig. 19); levofloxacin diluted at a concentration of 8 to 0.06 mug/ml (fig. 20); trimethoprim/Sulfamethoxazole (SXT) diluted at a concentration of 32 to 0.5 μ g/ml (FIG. 21); ciprofloxacin diluted at a concentration of 4 to 0.015 μ g/ml (fig. 22); and ceftriaxone diluted at a concentration of 64 μ g/ml to 0.12 μ g/ml (fig. 23).

As can be seen in FIGS. 16 to 23, E.coli (II)Escherichia coli) (QC strain, ATCC 25922) and clinically resistant E.coli (E.coli)E. coli) ("clinical") have similar MICs for imipenem, ceftazidime, gentamicin, levofloxacin, ciprofloxacin and ceftriaxone, whereas the two strains have different MICs for ampicillin and trimethoprim/Sulfamethoxazole (SXT). Thus, the data show clinically resistant E.coli: (E. coli) The strain is resistant to ampicillin and trimethoprim/Sulfamethoxazole (SXT). Thus, if a patient presents with an infection of this strain (or a similar strain), ampicillin and trimethoprim/Sulfamethoxazole (SXT) should not be administered; in contrast, imipenem, ceftazidime, gentamicin, levofloxacin, ciprofloxacin and ceftriaxone should be administered.

These data show that the present invention is able to accurately distinguish the MIC of an antimicrobial agent for a clinically relevant strain of bacteria that are resistant to one or more antimicrobial agents from the MIC of an antimicrobial agent for the same strain of bacteria that are susceptible to the antimicrobial agent; thus, the present invention can provide patients with an appropriate treatment regimen, i.e., a specific antimicrobial agent and at a specific dosage, in a substantially reduced amount of time relative to standard methods.

Example 8: the present invention provides a rapid and accurate determination of the MIC of various antimicrobial agents for antimicrobial sensitive bacteria.

In this example, the present invention was used to determine the susceptibility of various antimicrobial agents to antimicrobial agentsS. aureus) MIC of the strain.

Staphylococcus aureus (1)S. aureus) (QC strain 29213) in Mueller-Hinton (MH) liquid medium under standard sterile conditionsIncubated overnight at 37 ℃ with shaking. Staphylococcus aureus from overnight cultures was then transferred using McFarland standard techniques (S. aureus) The concentration is set to 5X 10 ⁵ CFU/ml, for optical density reading at 600 nm. At the same time, two sterile 96-well microplates were prepared with serial dilutions (both in MH broth) of the specific antimicrobial agent (see below) and the no antimicrobial agent (saline) control.

Microplates (each well containing 100 μ L) were inoculated with the prepared antimicrobial dilutions and incubated at 37 ℃ for 3 hours and 45 minutes for assays using the present invention ("rapid-AST" technique). After 3 hours and 45 minutes, the "quick-AST" microplates were removed from the shaking incubator and centrifuged at 2500 Xg for 2.5 minutes to pellet. The MH liquid medium was then aspirated and 100. Mu.L of 25 mM PBS was added to each well of both microplates. Then, 10 μ L of chemical moiety (here, europium-cryptate formulation) was added to each well (to 20 ng/well) and 10 μ L of 0.005% glutaraldehyde (as a signaling agent) was added to each well. Both microplates were then shaken at 300rpm for 30 minutes. After that, both plates were centrifuged at 2500 Xg for 2.5 minutes to pellet. The solution was aspirated and 200. Mu.L of PBS-tween wash was added to each well, followed by centrifugation to pellet. After aspirating the solution, a second identical wash of 200 μ L PBS-tween occurred, followed by final centrifugation to pellet. 200 μ L of PBS-tween was added to each well. Time resolved fluorescence plate readings were then used on a BioTek HI plate reader. The method was carried out with the following antimicrobial agent formulation: vancomycin diluted at a concentration of 32 to 0.25 μ g/ml (fig. 24); penicillin diluted at a concentration of 8 to 0.0625 μ g/ml (fig. 25); and teicoplanin diluted at a concentration of 16 to 0.0125 μ g/ml (fig. 26).

Staphylococcus aureus bacteria of the present invention (see FIGS. 24-26)S. aureus) The MIC determined (QC strain 29213) was similar to the MIC obtained from the standard CLSI reference method: vancomycin: 0.5-2 mug/ml; penicillin: 0.25-2 mug/ml; and teicoplanin 0.25-1 μ g/ml.

These data show that the present invention is capable of accurately determining the MIC of a variety of antimicrobial agents; thus, the present invention can provide patients with an appropriate treatment regimen, i.e., a particular antimicrobial agent and at a particular dosage, in a substantially reduced amount of time relative to standard methods.

Example 9: the present invention provides a rapid and accurate determination of the MIC of an antimicrobial agent directly from blood culture samples without the need for sub-culture and overnight growth incubations.

In this example, the invention was used to rapidly determine the MIC of an antimicrobial agent directly from a blood culture sample.

To Escherichia coli (A), (B) was obtainedE. coli) Staphylococcus aureus (1)S. aureus) And Klebsiella pneumoniae: (K. pneumoniae) One clinical sample each. The isolates were transferred on agar slants, sub-cultured, and stored at-80 ℃. The sample was removed from the freezer, allowed to warm to room temperature, and streaked on a 5% sheep blood-trypsin Treated Soy Agar (TSA) petri dish (ThermoFisher). The petri dishes were placed in an incubator at 35 ℃ overnight. Individual colonies were picked and the streaking process repeated on new plates followed by a second overnight incubation at 35 ℃. A total of three to five colonies were picked and dispersed into 1 mL sterile saline (Hardy Diagnostics) and concentration determined by optical density measurement (Molecular Devices M2) at 600 nm. Samples were diluted in two steps to 2 CFU/mL in 40mL sterile cation-conditioned Mueller Hinton liquid medium (MHB, hardy Diagnostics) in a capped flask.

The flasks were placed in a shake incubator overnight at 35 ℃ to simulate the performance of BD BACTEC blood culture system. After 10 hours the flask was placed at 4 ℃ at which point E.coli (E.coli)E. coli) The concentration was measured to be-1X 10 ⁸ CFU/ml. This is the approximate concentration at which commercial blood culture systems (such as BD BACTEC and bioMerieux Bact/Alert) record positive blood cultures. The 10 hour incubation time was determined by streaking blood culture samples onto 5% sheep blood TSA-petri dishes, incubating these overnight at 35 ℃, and determining the colony count.

Sub-culture "control" samples were collected by streaking the "positive" blood cultures onto TSA plates and incubating overnight at 35 ℃. Standard CLSI broth microdilution reference methods were then performed as described previously.

Centrifugation-based separation was then performed by following the SepsiTyper (Bruker Daltonics) protocol. Briefly, 1 mL lysis buffer (Bruker Daltonics) was added to 5 mL containing 1X10 ⁸ CFU/ml E.coli: (E. coli) MHB liquid medium. The mixture was aliquoted into six microcentrifuge tubes, vortexed for 10 seconds, and then spun at 13,000rpm for 2 minutes. The supernatant was removed and discarded, 1 mL wash buffer (Bruker) was added to each tube, and the tubes were centrifuged at 13,000rpm for 1 minute. The supernatant was removed again and discarded. Each pellet was resuspended in 500. Mu.L of sterile saline by pipetting up and down. The solutions were mixed and the bacterial concentration was determined using the Promega Bactitre-Glo ™ bacterial cell viability kit.

Sample at-5 × 10 ⁵ The concentration of CFU/ml was diluted into MHB. A "rapid-AST" assay (as described in example 2) was then performed and the MIC assays compared. The "rapid-AST" method on clinical samples provided MIC values similar to the standard method requiring sub-culture, see fig. 27 and 28.

These data show that the present invention ("rapid-AST" procedure), when used directly on clinical samples, provides results consistent with standard MIC-assay methods that require a sub-incubation step prior to overnight growth; thus, the present invention greatly reduces the time before an appropriate treatment regimen (i.e., a particular antimicrobial agent and at a particular dosage) is provided to a patient.

Example 10: europium-conjugated streptavidin binds to biotinylated wheat germ lectin, which specifically binds to gram-positive bacteria.

In this example, europium is used as the chemical moiety in a signaling agent comprising wheat germ agglutinin, which specifically binds gram positive bacteria.

Bacteria (Staphylococcus aureus: (A)S. aureus) In the range of 1x 10) ⁵ To 1x10 ⁹ Was seeded in MES buffer pH 6 across 96-well plates. Add 2. Mu.g of organisms to each well containing bacteria and the corresponding control wellThe reaction solution was incubated for 15 minutes to label the exterior of the bacteria in the wells with the selected reporter. Then, commercially available streptavidin-europium (e.g., from Perkin-Elmer) was added to a final concentration per well of 0.4. Mu.g/ml. After a further 15 minutes of incubation, the test plate was centrifuged at 2500 Xg for 2.5 minutes using Thermo Scientific Heraeus Multifuge X3 to pellet the bacteria at the bottom of the plate while leaving any irrelevant reporter in the supernatant. The plate was then pipetted using a BioTek Multiflo X plate washer to remove supernatant and unreacted reporter, followed by addition of wash buffer. This washing procedure was repeated two more times to completely remove any unreacted reporter. Finally, a reading buffer was added to the pipetted wells prior to the addition of the Delfia enhancing solution. The plates were then incubated for 15 minutes to allow europium enhancement before measuring it using time-resolved fluorescence on a BioTek HI plate reader, as shown in fig. 29.

These data show that using europium as the chemical moiety in the signaling agent enables accurate quantification of the bacterial concentration in solution.

Example 11: europium-conjugated streptavidin binds to biotinylated polymyxin B, which specifically binds to gram-negative bacteria.

In this example, europium is used as a chemical moiety in a signaling agent comprising polymyxin B, which specifically binds gram-negative bacteria.

Bacteria (Escherichia coli (E.coli) (ii))E coli) In the range of 1x 10) ⁵ To 1x10 ⁹ Was seeded in MES buffer pH 6 across 96-well plates. To each well containing bacteria and the corresponding control well was added biotinylated polymyxin (Hycult Biosciences) at a final dilution of 1:200 and the reaction solution is incubated for 15 minutes to facilitate labeling of the exterior of the bacteria within the wells with the selected reporter. Then, commercially available streptavidin-europium (e.g., from Perkin-Elmer) was added to a final concentration per well of 0.4. Mu.g/ml. After a further 15 minutes of incubation, the test plates were centrifuged at 2500 Xg for 2.5 minutes using Thermo Scientific Heraeus Multifuge X3So as to precipitate the bacteria at the bottom of the plate while leaving any irrelevant reporters in the supernatant. The plates were then pipetted using a BioTek Multiflo X plate washer to remove supernatant and unreacted reporter, followed by addition of wash buffer. This washing procedure was repeated two more times to completely remove any unreacted reporter. Finally, a reading buffer was added to the pipetted wells prior to the addition of the Delfia enhancing solution. The plates were then incubated for 15 minutes to allow europium enhancement before measuring it using time-resolved fluorescence on a BioTek HI plate reader, as shown in fig. 30.

These data show that using europium as the chemical moiety in the signaling agent enables accurate quantification of the bacterial concentration in solution.

Example 12: europium detectors provide a greater signal range and therefore more accurate MIC data.

This example compares the ability of europium and HRP as chemical moieties in a signaling agent (comprising an antibody that specifically binds to bacteria) to accurately determine the MIC.

Bacteria were prepared by diluting colonies into saline to achieve a McFarland value of 0.5 (which was verified using a spectrophotometer) using 96-well plates containing cationically conditioned Mueller Hinton broth and appropriate antimicrobial dilutions. It was diluted into saline at 1. The bacterial antimicrobial test plate was incubated at 35 ℃ and shaken at 150 rpm for 3 hours 45 minutes. After the incubation, the cationic magnetic beads and anti-staphylococcus aureus (S.aureus)S. aureus) Antibodies (conjugated with horseradish peroxidase or europium; custom conjugation by Cisbio Assays) was added to each well and incubated for 20 minutes. Using an automatic plate washer, the magnetic beads were captured and the contents of each well were washed three times with PBS-Tween20 (0.1%). Wells were then imaged directly using time-resolved fluorescence (europium), or TMB was added and allowed to incubate for 15 minutes, after which the reaction was stopped by adding 1M sulfuric acid and the absorbance at 450nm was measured for each well.

As shown in fig. 31, the MIC of SXT determined using europium as the chemical moiety was more accurate than the MIC determined using HRP as the chemical moiety.

These data show that the use of europium as a chemical moiety in a signaling agent enables accurate determination of the MIC of an antimicrobial agent.

Example 13. An embodiment of europium preparation that non-specifically labels bacteria is effective in detecting bacteria and quantifying bacterial concentration.

In this example, europium preparations bind non-specifically to bacteria.

Bacteria (Escherichia coli (E.coli) (ii))E coli) Across 96-well plates at concentrations ranging from 1e5 to 1e 9) in MES buffer (europium cryptate-diamine) or HEPES pH 7.5 (europium N1-amino) at pH 6. Europium cryptate-diamine (compound (3); cisbio) or europium N1-amino (compound (6); perkinelmer) was added at 66 ng/well to each well containing the bacteria and the corresponding control wells, followed by EDC/NHS (at 0.1 and 0.3 mg/ml) or glutaraldehyde (0.5% final concentration) as indicated.

Europium-cryptate-diamine (Eu-cryptate-diamine)

europium-N1-amino (Eu-N1-amino).

The reaction solution was incubated for 30 minutes to facilitate labeling of the exterior of the bacteria within the wells with the selected reporter. The test plate was then centrifuged at 2500 xg for 2.5 minutes using Thermo Scientific Heraeus Multifuge X3 to pellet the bacteria at the bottom of the plate while leaving any irrelevant reporter in the supernatant. The plates were then pipetted using a BioTek Multiflo X plate washer to remove supernatant and unreacted reporter, followed by addition of wash buffer. This washing procedure was repeated once more (Eu-cryptate-diamine) or twice (europium N1-amino) to completely remove any unreacted reporter. Wells containing europium cryptate-diamine were reconstituted in read buffer and read on a BioTek H1 plate reader using time resolved fluorescence as shown in figure 32. Finally, a read buffer was added to the aspirated wells treated with europium N1-amino prior to the addition of Delfia enhancing solution. The plates were then incubated for 15 minutes to allow europium enhancement before measuring it using time-resolved fluorescence on a BioTek HI plate reader, as shown in fig. 32.

These data show that europium preparations are able to accurately quantify the concentration of bacteria in solution when they bind non-specifically to bacteria.

Example 14 when quantifying bacterial concentration, europium can be attached to an amine via an isothiocyanate, or via NH ₂ Linked with carboxylic acids for non-specific labeling of bacteria.

In this example, europium preparations bind non-specifically to bacteria.

Medicine for curing pneumonia Klebsiella bacteria (A), (B), (C)Klebsiella pneumoniae) Or Escherichia coli (E coli) In the range of 1x10 ⁵ To 1x10 ⁹ Was seeded across 96-well plates in MES buffer (europium cryptate-diamine; compound (3)) or HEPES pH 7.5 (europium ITC; compound (4)) at pH 6. Europium cryptate-diamine (Cisbio) or europium ITC (Perkinelmer) was added at 66 ng/well to each well containing the bacteria and the corresponding control well, followed by EDC/NHS (at 0.1 and 0.3 mg/ml) to the wells containing the europium cryptate.

Europium-cryptate-diamine (Eu-cryptate-diamine)

Eu-N1-ITC (Delfia)。

The reaction solution was incubated for 30 minutes to facilitate labeling of the exterior of the bacteria within the wells with the selected reporter. The test plate was then centrifuged at 2500 xg for 2.5 minutes using Thermo Scientific Heraeus Multifuge X3 to pellet the bacteria at the bottom of the plate while leaving any irrelevant reporter in the supernatant. The plates were then pipetted using a BioTek Multiflo X plate washer to remove supernatant and unreacted reporter, followed by addition of wash buffer. This washing procedure was repeated once more (europium cryptate-diamine) or twice (PerkinElmer) to completely remove any unreacted reporter. Wells containing europium cryptate-diamine were reconstituted in read buffer and read on a BioTek H1 plate reader using time resolved fluorescence as shown in fig. 33. Finally, a reading buffer was added to the pipetted Eu-N1-treated wells before adding the Delfia enhancing solution. The plates were then incubated for 15 minutes to allow europium enhancement before measuring the europium using time-resolved fluorescence on a BioTek HI plate reader, as shown in fig. 33.

These data show that europium preparations are able to accurately quantify the concentration of bacteria in solution when they bind non-specifically to bacteria.

Example 15: glutaraldehyde can be used to non-specifically attach the europium-cryptate to the bacterial surface.

In this example, the europium preparation was non-specifically bound to the bacteria using glutaraldehyde.

Medicine for curing pneumonia Klebsiella bacteria (A), (B), (C)Klebsiella pneumoniae) Escherichia coli (E.coli)E coli) Or Staphylococcus aureus (Staph aureus) In the range of 1x10 ⁵ To 1x10 ⁹ Was seeded in MES buffer pH 6 across 96-well plates. To each well containing the bacteria and the corresponding control well was added 66 ng per well europium cryptate-diamine (compound (3); cisbio) followed by a 5% solution of glutaraldehyde to the wells containing the europium cryptate.

Europium-cryptate-diamine (Eu-cryptate-diamine).

The reaction solution was incubated for 30 minutes to facilitate labeling of the exterior of the bacteria within the wells with the selected reporter. The test plate was then centrifuged at 2500 xg for 2.5 minutes using Thermo Scientific Heraeus Multifuge X3 to pellet the bacteria at the bottom of the plate while leaving any irrelevant reporter in the supernatant. The plate was then pipetted using a BioTek Multiflo X plate washer to remove supernatant and unreacted reporter, followed by addition of wash buffer. This washing procedure was repeated once to completely remove any unreacted reporter. Wells containing europium cryptate-diamine were reconstituted in read buffer and read on a BioTek H1 plate reader using time resolved fluorescence as shown in fig. 34.

These data show that europium preparations are able to accurately quantify the concentration of bacteria in solution when they bind non-specifically to bacteria.

Example 16: EDC/NHS can be used to non-specifically couple europium-cryptates to bacterial surfaces.

In this example, europium preparations were non-specifically bound to bacteria using EDC/NHS.

Medicine for curing pneumonia Leberella bacteria (A), (B), (C)Klebsiella pneumoniae) Or Escherichia coli (E coli) In the range of 1x10 ⁵ To 1x10 ⁹ Was seeded in MES buffer pH 6 across 96-well plates. Europium cryptate-diamine (compound (3); cisbio) was added at 66 ng/well to each well containing the bacteria and the corresponding control well, followed by EDC/NHS (at 0.1 and 0.3 mg/ml) to the wells containing the europium cryptate.

Europium-cryptate-diamine (Eu-cryptate-diamine).

The reaction solution was incubated for 30 minutes to facilitate labeling of the exterior of the bacteria within the wells with the selected reporter. The test plate was then centrifuged at 2500 xg for 2.5 minutes using Thermo Scientific Heraeus Multifuge X3 to pellet the bacteria at the bottom of the plate while leaving any irrelevant reporter in the supernatant. The plate was then pipetted using a BioTek Multiflo X plate washer to remove supernatant and unreacted reporter, followed by addition of wash buffer. This washing procedure was repeated once to completely remove any unreacted reporter. Wells containing europium cryptate-diamine were reconstituted in read buffer and read on a BioTek H1 plate reader using time resolved fluorescence as shown in fig. 35.

These data show that europium preparations are able to accurately quantify the concentration of bacteria in solution when they bind non-specifically to the bacteria.

Example 17: effect of glutaraldehyde wash cycle on non-specific, cryptate-labeled bacteria.

In this example, europium preparations were non-specifically bound to bacteria using various washes containing glutaraldehyde.

Escherichia coli (A), (B) and (C)E coli) Or Staphylococcus aureus (Staph aureus) In the range of 1x10 ⁵ To 1x10 ⁹ Was seeded in MES buffer pH 6 across 96-well plates. Europium cryptate-diamine (compound (3); cisbio) was added to each well containing bacteria and the corresponding control well at 66 ng/well, followed by a 5% solution of glutaraldehyde to the wells containing the europium cryptate.

Europium-cryptate-diamine (Eu-cryptate-diamine).

The reaction solution was incubated for 30 minutes to facilitate labeling of the exterior of the bacteria within the wells with the selected reporter. The test plate was then centrifuged at 2500 xg for 2.5 minutes using Thermo Scientific Heraeus Multifuge X3 to pellet the bacteria at the bottom of the plate while leaving any irrelevant reporter in the supernatant. The plates were then pipetted using a BioTek Multiflo X plate washer to remove supernatant and unreacted reporter, followed by addition of wash buffer. This washing procedure was repeated twice to study the effect of multiple washes on the overall data quality. Wells containing europium cryptate-diamine were reconstituted in read buffer and read on a BioTek H1 plate reader using time resolved fluorescence as shown in fig. 36A-36C.

These data show that europium preparations can be non-specifically bound to bacteria using various washes containing glutaraldehyde.

Example 18: using NH ₂ The two-step labeling method of-PEG-biotin followed by streptavidin-europium (Eu-SAv) allows non-specific labeling of bacteria.

In this example, a two-step approach was used to non-specifically bind europium preparations to bacteria.

Escherichia coli (A), (B) and (C)E coli) In the range of 1x10 ⁵ To 1x10 ⁹ Was seeded in MES buffer pH 6 across 96-well plates. To each well containing the bacteria and the corresponding control well was added 1 mg/well of amine-PEG-biotin (Laysan Bio), followed by EDC/NHS (at 0.1 and 0.3 mg/ml) to the wells containing amine-PEG-biotin. The reaction solution was incubated for 15 minutes to facilitate external functionalization of the bacteria within the wells with biotin material. Streptavidin-europium (Eu-SAv) (Perkinelmer) was added to each reaction well at 400 ng/well. The reaction solution was incubated for 15 minutes to facilitate coupling between biotin and streptavidin. The test plate was then centrifuged at 2500 xg for 2.5 minutes using Thermo Scientific Heraeus Multifuge X3 to pellet the bacteria at the bottom of the plate while leaving any irrelevant reporter in the supernatant. The plates were then pipetted using a BioTek Multiflo X plate washer to remove supernatant and unreacted reporter, followed by addition of wash buffer. This washing procedure was repeated twice to study the effect of multiple washes on the overall data quality. Wells containing Eu-SAv were reconstituted in read buffer and read on a BioTek H1 plate reader using time resolved fluorescence as shown in fig. 37.

These data show that europium preparations can non-specifically bind bacteria using a two-step process comprising NH 2-PEG-biotin followed by Eu-SAv.

Example 19A two-step bacterial labeling procedure with NHS-LC-LC-Biotin followed by Eu-SAv allowed non-specific labeling of bacteria.

In this example, another two-step approach was used to non-specifically bind europium preparations to bacteria.

Escherichia coli (A), (B)E coli) In the range of 1x10 ⁵ To 1x10 ⁹ Was seeded in MES buffer pH 6 across 96-well plates. To each well containing bacteria and the corresponding control well was added 1 mg/well of amine-LC-LC-biotin (Thermo-Fisher) and then EDC/NHS (at 0.1 and 0.3 mg/ml) to the wells containing amine-PEG-biotin. The reaction solution was incubated for 15 minutes to facilitate external functionalization of the bacteria within the wells with biotin material. Streptavidin-europium (Perkin-Elmer) was added to each reaction well at 400 ng/well. The reaction solution was incubated for 15 minutes to facilitate coupling between biotin and streptavidin. The test plate was then centrifuged at 2500 xg for 2.5 minutes using Thermo Scientific Heraeus Multifuge X3 to pellet the bacteria at the bottom of the plate while leaving any irrelevant reporter in the supernatant. The plates were then pipetted using a BioTek Multiflo X plate washer to remove supernatant and unreacted reporter, followed by addition of wash buffer. This washing procedure was repeated twice to study the effect of multiple washes on the overall data quality. Wells containing europium cryptate-diamine were reconstituted in read buffer and read on a BioTek H1 plate reader using time resolved fluorescence as shown in fig. 38.

These data show that europium formulations can bind non-specifically to bacteria using a two-step process comprising NHS-LC-LC-biotin followed by Eu-SAv.

Example 20: filamentous bacteria can be isolated from solution using a filtration system with pores ranging in size from >0.2 microns to <10 microns; thereby providing more accurate chemosensitivity data.

This example illustrates an embodiment using filtration to exclude bacteria that have undergone filamentous growth in response to antimicrobial treatment.

Gram-negative rods in particular first undergo filamentous growth in response to sub-inhibitory concentrations of cell wall-acting antimicrobial agents (such as β -lactams). Although these will eventually be inhibited, the metabolic "volume" approach makes it significantly difficult to distinguish between antimicrobial resistant bacteria and bacteria that have undergone filamentous growth. When not considered, this filamentous growth incorrectly identifies the bacteria as more resistant than they actually are. The difficulty in determining antimicrobial resistance using the "volume" method is seen in fig. 39.

To avoid this, in one embodiment, each liquid culture medium microdilution is loaded into a filter comprising one or more predetermined pore sizes at the end of the incubation period (see fig. 40). The pore size is chosen such that a plurality of "normal" bacteria can pass through the filter, but capture filamentous bacteria greater than a certain length. The pore size may be >0.2 microns and <10 microns.

The filter can be designed for parallel sample processing, such as 96, 384 or 1586 well plates. A filter may be applied during the AST process as shown in fig. 40.

This example further illustrates the key advantage of designing a rapid AST platform that determines the presence of intact bacteria by surface area compared to traditional metabolic methods (which are essentially volumetric measurements).

Example 21: methods for making and using signaling agents comprising fluorescent nanoparticles.

In this example, methods for making and using signaling agents comprising fluorescent nanoparticles are described.

First, 20 mg fluorescein dilaurate (FL-DL) was weighed into a clear glass scintillation vial and 1000 mg ethanol was added. FL-DL was then dissolved in the vial via vortexing. Thereafter, 10 mg of DSPE-PEG-2 k-amine (Laysan Bio) was added to the mixture and dissolved by vortexing. Separately, 40 g deionized water was weighed into a beaker and added to a stir bar. The beaker was then placed on a magnetic stirrer and stirred at 200 RPM. Next, the ethanol solution was added to the beaker in a dropwise manner, and then the subsequent solution was introduced into the Microfluidics homogenizer and treated once at 6000 psi. 200 grams of deionized water was then added to the resulting mixture and the nanoparticles were purified and concentrated using Tangential Flow Filtration (TFF) from about 12-fold to 20 mL, then collected in glass scintillation vials. The collected nanoparticle preparation was filtered through a 0.2 μm filter and the nanoparticle size and concentration were determined by NanoSight (Malvern) with an average size reading of 102 nm.

The fluorophore-containing nanoparticles are functionalized with positively charged small molecules to form signaling agents that can be used in the present invention.

Escherichia coli (A), (B) and (C)Escherichia coli) (ATCC 11303) and ampicillin-resistant Escherichia coli (E.coli) ((ATCC 11303))E. coli) (ATCC 39936) was cultured in LB liquid medium at 37 ℃ under standard aseptic conditions. The concentration was determined by measuring the absorbance at 600nm (McFarland) and was set to 5X 10 by dilution ⁵ CFU/ml concentration. Ampicillin was then weighed into sterile water and added at the appropriate concentration to a sterile 3 mL microcentrifuge tube. The concentration is 8 x10 ⁸ Nanoparticles/ml of both bacteria and nanoparticles were added to these ampicillin coated sterile microcentrifuge tubes. The tubes were then capped and placed at 37 ℃ for 1.5 hours with continuous shaking, after which they were opened and each passed through a 0.2 μm filter. Next, 100 μ L of each filtrate was added to the wells of a 96-well plate, and 150 μ L of a chromogenic solution (5% tetramethylammonium hydroxide in ethanol) was added to each well. After 5 minutes, plates were read in a SpectraMax M2 microplate reader (Molecular Devices) at 490nm excitation/530 nm emission.

Fig. 41 shows the results of an assay using the nanoparticle-containing signaling agent prepared as described above. In this case, E.coli was treated with and without 100. Mu.g/ml ampicillin (at concentrations well above MIC) ((II))Escherichia coli) And ampicillin-resistant Escherichia coli (A), (B)E. coli). After removal of intact bacteria by filtration through a 0.2 μm filter, an assay for free signalling agents unrelated to intact bacteria was performed. Ampicillin-resistant E.coli treated with ampicillin, in which the ampicillin-free control group exhibited a low fluorescence signal (E. coli) As do the groups. Coli treated with ampicillin above the MIC showed a significant increase in fluorescence, indicating the efficacy of this antimicrobial agent.

FIG. 42 shows the results of E.coli measurements for various ampicillin concentrations. When ampicillin concentration is below MIC, -15 μ g/ml, the fluorescence signal is low and it rises at this value, indicating efficacy of the antimicrobial agent in this range.

Example 22: the present invention can be carried out using magnetic beads to isolate intact bacteria.

In this example, magnetic beads associated with reagents capable of binding intact bacteria are used to separate the intact bacteria from the solution.

According to the manufacturer's instructions (ThermoFisher), fromN1 μm beads activated with hydroxysuccinimidyl ester preparation of beads reactive with E.coli. Briefly, the beads were magnetically captured in supply and the storage solution was aspirated. The beads were then washed with ice cold 0.1M hydrochloric acid followed by addition of a polyclonal goat-anti-Lipopolysaccharide (LPS) antibody (Antibodies-Online inc.). The reaction was shaken for 2 hours according to the manufacturer's instructions, with vortexing every 5 minutes for the first 30 minutes. The beads were then washed thoroughly and stored in phosphate buffered saline, pH 7.4, at 4 ℃ until use.

The signaling agent comprises a moiety capable of binding to the microorganism (e.g., binding to E.coli: (E.coli) (E.coli))E. coli) The antibody of (e)) and a chemical moiety capable of providing a signal or contributing to the generation of a signal (e.g., horseradish peroxidase (HRP)).

Mixing anti-LPS magnetic beads and anti-Escherichia coli: (E. coli) The signal transduction reagent of (a) is simultaneously added to E.coli (E.coli) of a McFarland standard assayE. coli) In dilution series in MH liquid medium. The reaction was allowed to proceed for 20 minutes, followed by magnetic mounting in a 96-well microplate (V)&P Scientific) capture the magnetic beads. The wells were washed three times and then the detection solution described in example 1 was added. The optical densities at 450nm and 650nm were read for 10 minutes. The values at 5 minutes are plotted in fig. 43.

The procedure of example 1 was then used by adding a signaling agent and functionalized magnetic beads. Here, however, staphylococcus aureus (S.) (S. aureus) The strain was ATCC 12600, and three antimicrobial agents were used: ceftazidime, oxacillin and vancomycin.

After the incubation period, a solid having a size of 0.5 μm is added simultaneously with the signal transduction agentCationically charged magnetic beads (ChemiCell Fluidmag). The pH was adjusted to-8.4 by adding 50. Mu.L of borate buffer. The microplate was shaken on an orbital shaker for 20 minutes. The microplate was then placed on a magnetic capture plate containing 24 neodymium N52 magnetic materials. Then the MH liquid medium was aspirated and PBS containing 0.1% Tween-20 was added, and a total of 3 washes were performed. After the final aspiration, a stabilized chromogenic solution (ThermoFisher) consisting of 3,3',5,5' -Tetramethylbenzidine (TMB) and hydrogen peroxide was added and applied to a microplate reader (TM) ((TM))VmaxMolecular Devices) was monitored for 10 minutes at optical densities at 650nm and 450 nm. The data shown in fig. 44 depicts the 5 minute point from the beginning of incubation with the detection solution. As expected, increasing amounts of antimicrobial agent that caused lysis of bacterial cells reduced the number of intact bacteria.

These data show that functionalized magnetic particles can capture intact bacteria and enable quantification of intact bacteria (when bound to a signaling agent) after antimicrobial treatment. Such magnetic capture may be used in conjunction with or in lieu of other separation techniques to collect intact bacteria for use in the present invention.

Example 23: centrifugation of the bacterial solution provides a more accurate count of intact bacteria when compared to isolation of intact bacteria by functionalized magnetic beads.

In this example, the method for isolating intact bacteria using functionalized magnetic beads (as described in example 21 +) was compared to the method for isolating intact bacteria using centrifugation.

Capture of whole bacteria using functionalized magnetic beads was performed as described above. For centrifugation data, bacteria were washed three times by centrifugation at 2500 Xg for 2.5 minutes, manual aspiration, and addition of PBS-Tween. The magnetic beads were not used for centrifugal washing. Bacteria were treated with various concentrations of vancomycin ("VAN").

Figure 45 shows that centrifugation provides higher and more accurate bacterial numbers and MICs than separation by functionalized magnetic beads.

These data show that centrifugation of bacterial cultures for isolation of intact bacteria is superior to methods using functionalized magnetic particles. Separation using centrifugation can be used in conjunction with or in place of other separation techniques (e.g., magnetic separation) to collect intact bacteria for use in the present invention.

Example 24: chemical amplification via TAML nanoparticle amplifier allows 1x10 using standard optical detection equipment ³ To 1x10 ⁸ Signal transduction for optimal sensitivity in the CFU/ml range.

In this example, methods for making and using signaling agents comprising tetramino metal organoligand (TAML ®) catalysts are described.

Chemical amplification is achieved with proprietary nanoparticle amplifiers, which accommodate sophisticated nanoparticle formulation technology from drug delivery and small molecule catalysts from green chemistry. Each "nanomarker" comprises> >6×10 ⁴ A densely packed iron-containing tetraaminometal organic ligand (TAML) catalyst shielded by a polymer shell functionalized with specific ligands (FIG. 46). The molar activity of each TAML molecule was within 5-fold of that of horseradish peroxidase (gold standard immunoassay enzyme label) (fig. 46B). Upon specific binding, the nano-tag is chemically triggered to release its contents into solution, enabling homogeneous catalysis of the optical signal. The large number of catalysts per binding event enabled quantification of as few as 200 whole bacteria (fig. 46A) and a 100-fold sensitivity enhancement relative to standard enzyme immunoassays (fig. 46C). In addition to not requiring the development of new detection techniques, standard optical detection also enables compatibility with standard dry antimicrobial panel microplates (such as SensiTitre plates).

Claims (28)

1. A method for determining antimicrobial sensitivity of a microorganism, comprising:

incubating a liquid suspension of microorganisms in the presence of an antimicrobial agent under conditions that promote growth of the microorganisms;

adding a signaling agent capable of binding to the surface of the microorganism;

separating microorganisms bound by the signaling agent from unbound signaling agent; and

determining a level of signal associated with the microorganism as compared to one or more controls, thereby determining the antimicrobial sensitivity of the microorganism;

wherein the signaling agent comprises the structure:

wherein the microorganism is a bacterium.

2. The method of claim 1, wherein the antimicrobial sensitivity of the microorganism is determined in less than 5 hours.

3. The method of claim 1, wherein adding the signaling agent occurs after the incubating step.

4. The method of claim 1, wherein the signaling agent forms a non-covalent bond with the surface of the microorganism.

5. The method of claim 1, wherein the signaling agent forms a covalent bond with the surface of the microorganism in the presence of one or more agents that facilitate coupling, the reagent is selected from glutaraldehyde, formaldehyde, paraformaldehyde, EDC, DCC, CMC, DIC, HATU, wodwood's reagent,N,N' -carbonyldiimidazole, acrylates, amides, imides, anhydrides, chlorotriazines, epoxides, isocyanates, isothiocyanates, organic acids, monomers, polymers, silanes, silicates, NHS and sulfo-NHS or combinations thereof.

6. The method of claim 5, wherein the agent that facilitates coupling is glutaraldehyde.

7. The method of claim 1, wherein multiple antimicrobial agents are tested in parallel.

8. The method of claim 1, wherein said determining a signal level comprises measuring a signal level associated with a microorganism.

9. The method of claim 8, wherein the method further comprises the steps of: determining whether the microorganism is resistant, moderately resistant, or sensitive to the one or more antimicrobial agents and/or determining the Minimum Inhibitory Concentration (MIC) of the one or more antimicrobial agents against the microorganism based on the level of signal associated with the intact microorganism.

10. The method of claim 1, wherein the method does not involve the step of capturing the microorganism on the solid surface before or during the incubation and/or does not include the step of growing the microorganism on the solid surface before or after the incubation step.

11. The method of claim 1, wherein the separation of the microorganisms is performed by centrifugation, magnetic separation, filtration, electrophoresis, dielectrophoresis, precipitation, agglutination, or a combination thereof.

12. The method of claim 1, wherein the one or more controls comprise a positive control measured from a microorganism under otherwise identical conditions but without the antimicrobial agent or with the antimicrobial agent to which the one or more microorganisms are not susceptible.

13. The method of claim 1, wherein the microorganism is obtained from a biological sample from a subject having an infection with the microorganism and/or from a culture derived from the biological sample.

14. The method of claim 13, wherein the biological sample is selected from the group consisting of blood or a blood component, bronchoalveolar lavage, cerebrospinal fluid, nasal swab, sputum, stool, throat swab, vaginal swab, urine, and wound swab, or a combination thereof.

15. The method of claim 1, wherein the steps of incubating the liquid suspension of microorganisms and adding a signaling agent occur in a cartridge comprising a plurality of chambers and the step of determining a level of signal associated with a microorganism comprises determining a level of signaling in the plurality of chambers.

16. The method of claim 15, wherein the cartridge further comprises one or more control chambers that do not contain an antimicrobial agent or an antimicrobial agent to which one or more microorganisms are not sensitive.

17. A method for determining antimicrobial sensitivity of a microorganism, comprising:

incubating a liquid suspension of a microorganism in the presence of an antimicrobial agent and a signaling agent under conditions that promote growth of the microorganism, wherein the signaling agent is capable of binding to the surface of the microorganism;

separating microorganisms bound by the signaling agent from unbound signaling agent; and

determining a level of signal associated with the microorganism as compared to one or more controls, thereby determining antimicrobial sensitivity of the microorganism;

wherein the signaling agent comprises the structure:

wherein the microorganism is a bacterium.

18. The method of claim 17, wherein the antimicrobial sensitivity of the microorganism is determined in less than 5 hours.

19. The method of claim 17, wherein the one or more controls comprise a positive control measured from a microorganism under otherwise identical conditions, but without the antimicrobial agent or with the antimicrobial agent to which the one or more microorganisms are not susceptible.

20. The method of claim 17, wherein the signaling agent forms a non-covalent bond with the surface of the microorganism.

21. The method of claim 17, wherein the signaling agent forms a covalent bond with the surface of the microorganism in the presence of one or more agents that facilitate coupling, the reagent is selected from glutaraldehyde, formaldehyde, paraformaldehyde, EDC, DCC, CMC, DIC, HATU, wodwood's reagent,N,N' -carbonyldiimidazole, acrylates, amides, imides, anhydrides, chlorotriazines, epoxides, isocyanates, isothiocyanates, organic acids, monomers, polymers, silanes, silicates, NHS and sulfo-NHS or combinations thereof.

22. The method of claim 21, wherein the agent that facilitates coupling is glutaraldehyde.

23. The method of claim 17, wherein the microorganism is obtained from a biological sample from a subject having an infection with the microorganism and/or from a culture derived from the biological sample; and wherein the biological sample is selected from the group consisting of blood or a blood component, bronchoalveolar lavage, cerebrospinal fluid, a nasal swab, sputum, stool, a throat swab, a vaginal swab, urine, and a wound swab, or a combination thereof.

24. A method for determining antimicrobial sensitivity of a microorganism, comprising:

incubating a liquid suspension of microorganisms in a cartridge comprising a plurality of chambers, each chamber containing one or more antimicrobial agents, under conditions that promote growth of the microorganisms;

adding a signaling agent to the plurality of chambers, wherein the signaling agent is capable of binding to a surface of a microorganism;

removing unbound signaling agent; and

determining the level of signaling in the plurality of chambers as compared to one or more controls, thereby determining the sensitivity of the microorganism to the one or more antimicrobial agents;

wherein the signaling agent comprises the structure:

wherein the microorganism is a bacterium.

25. The method of claim 24, wherein the antimicrobial sensitivity of the microorganism is determined in less than 5 hours.

26. The method of claim 24, wherein the cartridge further comprises one or more control compartments that do not contain an antimicrobial agent or an antimicrobial agent to which one or more microorganisms are not sensitive.

27. The method of claim 24, wherein the signaling agent forms a covalent bond with the surface of the microorganism in the presence of one or more agents that facilitate coupling, the reagent is selected from glutaraldehyde, formaldehyde, paraformaldehyde, EDC, DCC, CMC, DIC, HATU, wodwood's reagent,N,N' -carbonyldiimidazole, acrylates, amides, imides, anhydrides, chlorotriazines, epoxides, isocyanates, isothiocyanates, organic acids, monomers, polymers, silanes, silicates, NHS and sulfo-NHS or combinations thereof.

28. The method of claim 24, wherein the microorganism is obtained from a biological sample from a subject having an infection with the microorganism and/or from a culture derived from the biological sample; and wherein the biological sample is selected from the group consisting of blood or a blood component, bronchoalveolar lavage, cerebrospinal fluid, a nasal swab, sputum, stool, a throat swab, a vaginal swab, urine, and a wound swab, or a combination thereof.

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